U.S. patent application number 14/439330 was filed with the patent office on 2015-12-17 for novel reagent for gene-drug therapeutics.
The applicant listed for this patent is AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH, NATIONAL UNIVERSITY OF SINGAPORE. Invention is credited to Yoon Khei Ho, Heng-Phon Too, Lihan Zhou.
Application Number | 20150361449 14/439330 |
Document ID | / |
Family ID | 50627830 |
Filed Date | 2015-12-17 |
United States Patent
Application |
20150361449 |
Kind Code |
A1 |
Too; Heng-Phon ; et
al. |
December 17, 2015 |
NOVEL REAGENT FOR GENE-DRUG THERAPEUTICS
Abstract
The present invention relates to a composition for transfecting
a cell with a genetic material comprising a first agent capable of
directing the genetic material away from the acidic compartments in
the cell and a second agent capable of stabilizing the microtubule
or a network thereof. The invention also relates to the use of the
composition in the manufacture of a medicament for treating a
disease, a method for delivering a genetic material into a cell and
a kit.
Inventors: |
Too; Heng-Phon; (Singapore,
SG) ; Ho; Yoon Khei; (Singapore, SG) ; Zhou;
Lihan; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH
NATIONAL UNIVERSITY OF SINGAPORE |
Singapore
Singapore |
|
SG
SG |
|
|
Family ID: |
50627830 |
Appl. No.: |
14/439330 |
Filed: |
October 29, 2013 |
PCT Filed: |
October 29, 2013 |
PCT NO: |
PCT/SG2013/000464 |
371 Date: |
April 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61719908 |
Oct 29, 2012 |
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Current U.S.
Class: |
435/455 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 31/711 20130101; A61K 48/00 20130101; A61K 39/0011 20130101;
A61P 43/00 20180101; A61K 2039/5156 20130101; C12N 15/85 20130101;
A61K 2039/55522 20130101; A61P 35/00 20180101; A61K 47/34 20130101;
A61K 2039/5152 20130101; C12N 15/87 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85 |
Claims
1.-39. (canceled)
40. An in vitro method for enhancing intracellular trafficking of
genetic material in a cell, the method comprising: a) transfecting
the cell with the genetic material, b) adding a first agent and a
second agent simultaneously, separately or sequentially to the cell
after transfection, wherein the first agent directs the genetic
material away from the acidic compartments in the cell, and wherein
the second agent stabilises the microtubule or a network thereof,
wherein the first agent is a lipid and/or peptide fusogenic agent,
and wherein the second agent is selected from a group consisting of
a histone deacetylase inhibitor (HDACi), a tubulin binding agent
(TBA), a siRNA that directly or indirectly affects the microtubule
network stability, and combinations thereof.
41. The method according to claim 40, wherein the lipid fusogenic
agent is selected from the group consisting of DOPE, CHEMS, DPPC
and DOPC and combinations thereof.
42. The method according to claim 40, wherein the peptide fusogenic
agent is selected from the group consisting of haemagglutinin
(HA2-peptide), influenza-derived fusogenic peptide diINF-7, and T
domain of Diphtheria toxin, polycationic peptides, polylysine,
polyarginine, and combinations thereof; or wherein the peptide
fusogenic agent is chemically modified by an attachment selected
from the group comprising of biomolecules, lipids, nucleic acids
and synthetic carriers.
43. The method according to claim 40, wherein the second agent
enhances tubulin acetylation; or wherein the second agent enhances
the sensitivity of the cell to a therapeutic agent and/or modifies
the host genetic status.
44. The method according to claim 40, wherein the HDACi is selected
from the group consisting of Tubastatin A, belinostat, bufexamac,
panobinostat, PCI-24781, SAHA (vorinostat), scriptaid, trichostatin
A, valproic acid, salermide, sirtinol, and combinations
thereof.
45. The method according to claim 40, wherein the TBA is selected
from the group consisting of taxanes, epothilones, and a
combination thereof;
46. The method according to claim 45, wherein the taxanes are
selected from the group consisting of paclitaxel, docetaxel, and a
combination thereof; and wherein the epothilones are selected from
the group consisting of patupilone, ixabepilone, BMS 310705,
sagopilone, KOS-862, KOS-1584, and combinations thereof.
47. The method according to claim 40, wherein the genetic material
is coupled to at least one cationic species.
48. The method according to claim 47, wherein the cationic species
is selected from the group consisting of polyethylene imine,
polycationic amphiphiles, DEAE-dextran, cationic polymers, their
derivatives, and combinations thereof.
49. The method according to claim 48, wherein the cationic species
is cationic polymers selected from the group consisting of
dendimers, branched-polyethylenimine (BPEI),
linear-polyethylenimine (LPEI), Poly(amidoamine) (PAMAM),
XtremeGENE XPHP.RTM., and combinations thereof.
50. The method according to claim 47, wherein the nucleic acid:
polymer (N/P) ratio of genetic material to cationic species is
selected from the group consisting of about 0 to about 1000, about
0 to about 500, about 0 to about 100, about 0 to about 50 and about
0 to about 20; and wherein the N/P ratio is the ratio of the
genetic material to the cationic species forming polyplexes within
the composition.
51. The method according to claim 40, wherein the genetic material
is selected from the group consisting of DNA, RNA, mRNA, ribozymes,
antisense oligonucleotides, modified polynucleotides and
combinations thereof.
52. The method according to claim 40, wherein the cell is a
differentiated or undifferentiated cell.
53. The method according to claim 40, wherein the cell is selected
from the group consisting of nervous systems cell, liver cell,
hematopoiesis cell, peripheral blood cell, umbilical blood cell,
bone marrow cell, tumour cell, ischemic tissue cell, skin cells,
stem cells, cancer cell lines and combinations thereof.
54. The method according to claim 53, wherein the cancer cell lines
are selected from the group consisting of U87MG (ATCC no. HTB-14),
U251MG (CLS ID no. 300385), MCF7 (ATCC no. HTB-22), MDA-MB-231
(ATCC no. HTB-26), MCF10a (ATCC no. CLR-10317), RAW 264.7 (ATCC no.
TIB-71), PC3 (ATCC no. CRL-1435), M14 (Pubmed ID:12354931), MEF
(ATCC no. SCRC-1040), Neuro2A (ATCC no. CCL-131), NG-108 (ATCC no.
HB-12317) and HeLa (ATCC no. CCL-2).
55. The method according to claim 40, wherein the first agent and
the second agent are provided within the first hour or within 10
hours of transfection of the cell with the genetic material.
56. The method according to claim 55, wherein the second agent is
to be provided after the first agent.
57. The method according to claim 40 for use in gene therapy.
58. The method according to claim 40, further comprising a
therapeutic agent.
59. The method according to claim 58, wherein the therapeutic agent
is a chemotherapeutic agent selected from the group consisting of
paclitaxel, docetaxel and epothilones, and combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
provisional application No. 61/719,908 filed Oct. 29 2012, the
contents of it being hereby incorporated by reference in its
entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present disclosure relates to composition for
transfecting a cell with a genetic material. In particular, the
invention relates to transfecting a cell using comprising a first
agent capable of directing the genetic material away from the
acidic compartments in the cell and a second agent capable of
stabilizing the microtubule or a network thereof.
BACKGROUND OF THE INVENTION
[0003] Gene delivery of pDNA, antisense oligonucleotides and shRNA
offers the potential for the treatment of devastating disorders
including neurodegenerative diseases and spinal cord injuries, for
which there are currently few treatment options. However, nucleic
acid-based therapeutics is still in the early stages of development
as a new category of biologics. The efficacy of gene delivery
requires delivery of these molecules to the interior of the cell,
presenting significant challenges for delivery strategies. Given
the disadvantages associated with clinical application of viral
carriers, as existing non-viral carriers transfect differentiated
neurons poorly, non-viral gene delivery serves as an attractive
alternative due to reduced immunogenicity, the ability to
accommodate large size of transgenes, improved safety, and ease of
manufacturing.
[0004] Polyethylenimine (LPEI), an off-the-shelf transfection
agent, has been used to transfect a variety of cell types,
including neurons in vivo and in vitro. It is thought that LPEI
condenses DNA into nanoparticles, along with the cationic property
of LPEI, facilitates entry of these vectors into cells by binding
to negatively charged heparan sulfate proteoglycans on the cell
surface. Following internalization, LPEI/DNA nanocomplexes are
thought to be transported to the perinuclear region of cells. LPEI
is then hypothesized to escape endosomes through a "proton-sponge
effect", releasing the DNA from the polymer and the DNA
subsequently taken up into the nucleus.
[0005] Recent efforts to increase transfection efficiency and cell
viability of differentiated neuron by optimizing protocols using
LPEI have met with limited success. Attempts have been made to
identify underlying mechanisms limiting high transfection
efficiency in non-neuronal cells, primary neurons and neuronal
cell-lines. Poor transfection of non-dividing, post-mitotic cells
including neuronal cells is often thought to be due to the presumed
inability of the pDNA in nuclear translocation. In addition,
internalization and intracellular barriers were thought to restrict
transfection in differentiated neuronal cell. By increasing uptake,
more neuronal cells were found to express transgene, but only
marginally (from 2% to 6% of total cell population). To date, the
goal of attaining high transfection efficiency in differentiated
neuron using non-viral carriers remains elusive and efforts to
produce even more novel polymers with such properties
continues.
[0006] Despite significant improvements in diagnosis and
innovations in the treatment for various devastating diseases, such
as cancer, autoimmune disease, and neurodegenerative disease,
effective treatment of these disorders still presents major
challenges. Presently, low transfection and delivery efficiencies
limit the application of drug-gene therapeutics. It is this unmet
need that requires the development of methods to enhance gene
delivery ex vivo and in vivo and the subsequent development of
galenics using this technology.
[0007] Accordingly, it is an aim of the present disclosure to
ameliorate the above-mentioned disadvantages and provide an
improving transfection efficiency.
SUMMARY OF THE INVENTION
[0008] In a first aspect, the present invention provides a
composition for transfecting a cell with a genetic material
comprising a first agent capable of directing the genetic material
away from an acidic compartment in the cell and a second agent
capable of stabilizing the microtubule or a network thereof.
[0009] In a second aspect, the present invention provides a method
of delivering a genetic material into a cell comprising the step of
administering the genetic material with a composition.
[0010] In a third aspect, the present invention provide a use of a
composition in the manufacture of a medicament for treating a
disease, selected from a group consisting of cancer, SMA, bone
cancers, leukemia, blood cancers, sickle cell disease,
Wiskott-Aldrich Syndrome, HIV, genetic disease, diabetes, cardiac
disease and neurodegenerative diseases
[0011] In a fourth aspect, there is provided a composition
comprising a first agent, as described herein, and a second agent,
as described herein.
[0012] In a fifth aspect, the present invention provides a kit
according comprising a composition as described herein and
instructions for use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The invention will be better understood with reference to
the detailed description when considered in conjunction with the
non-limiting examples and the accompanying drawings, in which:
[0014] FIG. 1 shows a schematic of the possible effects of
TrafEn.TM.. It contains an optimized mix of chemoRe-router that
specifically re-directs polyplexes away from the acidic compartment
(step 1) and re-routing onto microtubular networks stabilized by
the use of chemosensitizers (step 2) (e.g., histone deacetylase
inhibitor (HDACi), taxanes). The chemosensitizers synergize with
the gene product to achieve beneficial therapeutic effects (step
3). TrafEn.TM. may also be used to improve intracellular
trafficking of therapeutic material.
[0015] FIG. 2 shows a schematic of the composition of TrafEn.TM.,
which contains an optimized mix of chemoRe-router and microtubule
targeting chemosensitizer. TrafEn.TM. is shown to enhance
transfection efficiency and synergize\ with the gene product to
achieve beneficial therapeutic effects.
[0016] FIG. 3 shows the effects of a microtubular network modifier.
(A) shows that microtubule dynamicity is inhibited by tubulin
acetylation or tubulin binding agents (TBA). TBA, such as
chemotherapeutic taxanes, binds to tubulin while inhibition of
HDAC6 and Sirtuin2 prevents deacetylations of microtubules; leading
to microtubule stabilization. (B) depicts the possible effects of
histone deacetylation inhibitors (HDACi). These appear to ease the
pathological conditions of heart failure via possible multiple
mechanisms, including derepression of protective genes and
inhibition of the expression of pro-inflammatory genes. Other
non-transcriptional effects of HDACi may include the enhancement of
acetylation of sacromeric proteins and the inhibition of pathways
for autophagy and apoptosis.
[0017] FIG. 4 shows a schematic depicting the synergistic effect of
TrafEn.TM. and its use as a novel gene-drug therapy combinatorial
therapeutic. As described previously in FIG. 2, the components that
make up TrafEn.TM. are a microtubule targeting chemosensitizer and
a chemoRe-router. Both these components interact to aid and enhance
a gene transfer process, which could result in an up- or
down-regulation of gene expression of a targeted gene. This
regulation of gene expression, together with the chemosensitizer,
may result in a desired therapeutic effect, which could be, but is
not limited to, cell reprogramming, stem cell differentiation,
pro-apoptotic effect, anti-angiogenic effect, anti-inflammatory
effect and/or a neuroprotective effect.
[0018] FIG. 5 shows data that aggregated polyplex transfected
native, but not differentiated neuronal cells efficiently. A. Cells
were differentiated by 50 ng/ml of GDNF, 10 .mu.M of all trans
Retinoic acid (RA), or 10 .mu.M Forskolin (FSK) for 48 h prior to
transfection. Native and differentiated cells were transfected with
polyplex (N/P=20) using mild centrifugation. Percentage of cells
expressing green fluorescence protein (EGFP) was quantified by FACS
48 h post transfection. B. Representative fluorescent images
(bottom rows), acquired 48 h post transfection. C. Percentages of
EGFP positive (+) and negative (-) Neuro2A cells (native and
differentiated phenotypes) treated with 10 .mu.M RA were determined
by counting fluorescent and bright field images. Cells bearing
neurites twice the cell body length were considered as
differentiated phenotype. The data shown were the mean.+-.s.e.m.,
n=4.
[0019] FIG. 6 shows micrographs and histograms showing that the
internalization of polyplex was unaffected after differentiation.
A. Native and differentiated Neuro2A and NG-108 cells were
transfected with Rhodamine-pDNA polyplex (N/P=20) using mild
centrifugation procedure. After 4 h incubation, cells images were
taken before and after quenching with 0.4% trypan blue. Bar
represents 10 .mu.m. B. At various time points post transfection by
polyplex (N/P=20), cells were treated with pAA/DNAse to remove
extracellular pDNA. Cells were trypsinized and treated with
pAA/urea lysis buffer before quantification of internalized DNA by
qPCR. The data shown were the mean.+-.s.e.m., n=3.
[0020] FIG. 7 shows data showing that PKC was involved in the
uptake of polyplex in native but not differentiated neuronal cells.
A. Native and differentiated (10 .mu.M RA) Neuro2a was treated with
2.5 .mu.g/ml rottlerin, 5 .mu.g/ml Filipin III or 30 .mu.g/ml
Dynasore for 45 min prior transfection. DMSO/0.5% FBS was used as
control for treatment. After transfection with polyplex (N/P=20),
cells were incubated at 4.degree. C. or 37.degree. C. for 4 h.
Extracellular pDNA was removed by pAA/DNAse and cells were
trypsinized. Then, samples were treated with pAA/urea lysis buffer
and the absolute copy number of pDNA was quantified by qPCR. In
similar experiments, native Neuro2A cells were incubated at
37.degree. C. for 24 h post transfection. B. Representative
fluorescent images (bottom rows), acquired 48 h post transfection.
C. Percentage of cells expressing EGFP was counted. Significant
differences in transfection efficiencies were calculated using the
two tailed student's t-test. The data shown were the
mean.+-.s.e.m., n=4. *, p<0.05; **, p<0.005.
[0021] FIG. 8 shows micrograph images and column graphs, showing
that polyplex localized differentially in native and differentiated
neuronal cells. A. Native and differentiated (10 .mu.M RA) Neuro2A
cells were transfected using LPEI/FITC-pDNA (N/P=20) with
centrifugation transfection procedure. Quenching reagent EtBr (20
.mu.g/ml) was added 4 h post transfection. Cell images were taken
before and after quenching by EtBr. Bar represents 5 .mu.m. B.
Cells were transfected by pre-complexed LPEI/FITC- or
Rhodamine-pDNA at N/P=20. Percentage of cells associated with
labeled pDNA was acquired after quenching of extracellular
fluorescence with EtBr or trypan blue, at 4 or 24 h post
transfection. Ratios of FITC/Rhodamine (FITC/Rho) in native and
differentiated Neuro2A cells were calculated. C. Four hour after
transfection of native and differentiated Neuro2A with
LPEI/Rhodamine-pDNA (N/P=20), culture media was removed and
replenished with 1.times.PBS containing 50 nM Lysotracker green
DND-26 and the incubation continued for 5 min before visualization
using confocal microscopy. Images of single cell were captured at
100.times. magnification. Asterisks indicated co-localization of
polyplex with the labelled compartments. D. Co-localized pixels of
Rhodamine with lysotracker green DND-26 was analysed. The data
shown were the mean.+-.s.e.m (n=20).
[0022] FIG. 9 visualises data showing that endosomal escape of
polyplex, facilitated by DOPE/CHEMS, greatly enhanced transfection.
A. Neuro2A cells differentiated with RA were transfected by
LPEI/pDNA at N/P=20 with centrifugation transfection procedure.
Pre-complexed DOPE/CHEMS (9:2 molar ratio) in HEPES was added to
the culture medium immediately post transfection. Transfection
efficiency was analysed by counting fluorescent and bright field
images 24 h later. Percentage of EGFP positive cells bearing
neurites twice the cell body length was presented as group
mean.+-.s.e.m (n=4). B. Representative images captured at the end
of incubation were presented. C. Native and differentiated Neuro2A
cells were transfected by LPEURhodamine-pDNA (N/P=20). Then, acidic
compartment was labelled with lysotracker green at 4 h post
transfection. Images of single cell were captured at 100.times.
magnification and D. the co-localized pixels were analysed. Data
were presented as group mean.+-.s.e.m (n=20).
[0023] FIG. 10 shows histograms and micrographs, showing data that
enhanced trafficking following endosomal escape of polyplex greatly
improved transfection. Native and differentiated (10 .mu.M RA)
Neuro2A cells or primary cortical neurons (DIV 3) were transfected
with LPEI/pDNA (N/P=20) using centrifugation transfection
procedure. A. For differentiated Neuro2A, in the presence/absence
of DOPE/CHEMS, Tubastatin A (5 .mu.M) was added 1 h post
transfection and further incubated for 24 h. For native Neuro2A
(black bar), the whole cell population was counted. For
differentiated Neuro2A (grey bar), cells bearing neurites twice the
cell body length were counted. Data presented as mean.+-.s.e.m
(n=4). Representative images (20.times. magnification) of
transfected differentiated cells (left panel). B. Primary cortical
neurons were initially exposed to the polyplex and subsequently
with neurobasal media containing the various combinations of
DOPE/CHEMS and Tubastatin A (16 .mu.M). The treatment was
terminated 24 h later and the cells were incubated for a further 48
h in fresh neurobasal media. Transfection efficiency was quantified
by FACS and represented as mean.+-.s.d (n=3). Representative images
(20.times. magnification) were shown. Significant differences in
transfection efficiencies were calculated using the two tailed
student's t-test. *, p<0.05; **, p<0.005.
[0024] FIG. 11 shows histograms visualising that increasing N/P
ratios reduced cell viability. Native and differentiated A. Neuro2A
and B. NG-108 cells were transfected with pIRES-EGFP-EV71 complexed
with LPEI at various N/P ratios. In a bolus transfection, the cells
were exposed to transfection mixture for 4 h. Cell viability was
measured by MTS assay after 48 h incubation in fresh media. Data
points were expressed as a percentage of control (N/P=0) and group
mean.+-.s.e.m. (n=6). Significant differences in cell viabilities
against control (N/P=0) were calculated using the two tailed
student's t-test. *, p<0.05; **, p<0.005.
[0025] FIG. 12 shows histograms depicting that mild centrifugation
improved transfection efficiency. Neuro2A cells were transfected
with LPEI/pDNA using A. N/P=20 and B. various N/P ratios over
different periods of time, with or without centrifugation at the
end of incubation. Percentage of EGFP positive cells was quantified
by FACS 48 h post transfection. The data shown are the
mean.+-.s.e.m., n=4.
[0026] FIG. 13 shows, using a scatter plot and micrographs, that
polyplexes aggregated and deposited in DMEM. A. Kinetic analysis of
polyplex (N/P=20) size growth in HEPES or DMEM as measured by
Dynamic Light Scattering over a period of 25 min. B. LPEI/FITC-pDNA
(N/P=20) was prepared and incubated in either HEPES or, DMEM for 15
min. In the absence or presence of Neuro2A cells, the transfection
mixtures were centrifuged. Representative merged images of DIC and
fluorescence were presented. Bars represent 10 .mu.M.
[0027] FIG. 14 shows micrograph depicting data that mild
centrifugation and short incubation durations resulted in high
transfection efficiency at low toxicity. Native and differentiated
A. Neuro2A and B. NG-108 cells were transfected with LPEI/pDNA at
various N/P ratios using the centrifugation transfection procedure.
Cell viability and transfection efficiency was measured 48 h post
transfection using cell viability assay and FACS, respectively.
Transfection efficiencies and cell viability shown were the
mean.+-.s.e.m. (n=4) and mean.+-.s.e.m. (n=6) respectively.
[0028] FIG. 15 shows column graphs and an image of a gel
electrophoresis that pAA/urea lysis buffer efficiently dissociated
polyplexes. A. Plasmid (10.sup.6 copies) in different solutions
(water, pAA, urea lysis buffer and pAA/urea lysis buffer) was
quantified by qPCR and normalized to control (pDNA in water). The
lysis solution used did not affect the amplification efficiency of
qPCR. LPEI/pDNA at different N/P ratios was treated with pAA/urea
lysis buffer or water at 95.degree. C. for 30 min. Subsequently,
samples were analysed by B. gel retardation assay and C. qPCR. Data
shown were mean.+-.s.e.m. (n=3).
[0029] FIG. 16 shows histograms showing that the cellular binding
of polyplexes was not affected after neuronal differentiation. A.
Neuro2A and B. NG-108 were transfected by LPEI/pDNA (N/P=20) via
centrifugation transfection procedure. Cells were harvested at
different time points post transfection and treated with pAA/urea
lysis buffer. Absolute copy number of pDNA associated with cells
was quantified using realtime qPCR. The data shown were the
mean.+-.s.e.m., n=3.
[0030] FIG. 17 shows bright field images that trypan blue
efficiently quenched surface bound LPEI-Rho labelled DNA complexes.
LPEI/Rhodamine-pDNA complexes (N/P=20) were first deposited onto
Neuro2A cells. Next, transfection mixture was replaced with iced
cold complete media and further incubated for 4 h at 4.degree. C.
Then, trypan blue was added. Cell images were taken before and
after quenching by 0.4% trypan blue. As internalization is
inhibited at 4.degree. C., the surface bound fluorescent labelled
DNA was efficiently quenched. Bar represents 20 .mu.m.
[0031] FIG. 18 Data shown as column graphs and fluorescent images
show that DNAse efficiently removed surface bound polyplexes. A.
LPEI/pDNA (N/P=20) pre-complexed in DMEM was deposited on Neuro2A
cells after centrifugation. Cells were incubated at 4.degree. C. or
37.degree. C. for 4 h and treated with pAA/DNase in DMEM to remove
extracellular pDNA. Two hours later, cells were harvested by
trypsinization and treated with pAA/urea lysis buffer before
quantification of internalized pDNA using qPCR. The data shown were
the mean.+-.s.e.m., n=4. B. After deposition of polyplexes, wells
were washed once with PBS and replenished with (DMEM with or
without pAA/DNAse). Surface bound polyplexes were visualized by
Sybr Green I staining in 1.times.PBS and incubated for 5 min. After
1.times. wash with PBS, images were captured at 10.times.
magnification. Representative images were shown.
[0032] FIG. 19 shows fluorescent images showing that PKC inhibitor
substantially reduced transfection. Neuro2A cells were treated with
GO6983 (2 .mu.M) for 45 min prior transfection. Transfection
mixture (LPEI/pDNA at N/P=20) was replaced with DMEM containing
0.5% FBS and GO6983 (2 .mu.M). Representative images (acquired 24 h
post transfection) were shown.
[0033] FIG. 20 visualises data, here as histograms and fluorescent
images that shows that PKC involved in the uptake of polyplexes in
native but not differentiated neuronal cells. A. Native and
differentiated (10 .mu.M FSK) NG-108 was treated with rottlerin,
Filipin III or Dynasore for 45 min prior transfection. DMSO/0.5%
FBS was used as control for treatment. After transfection
(LPEi/pDNA at N/P=20), cells were incubated at 4.degree. C. or
37.degree. C. for 4 h. Extracellular pDNA was removed by pAA/DNAse
and cells were trypsinized. Then, samples were treated with
pAA/urea lysis buffer and absolute copy number of pDNA was
quantified by qPCR. In similar experiments, native NG-108 cells
were incubated at 37.degree. C. for 24 h post transfection. B.
Representative images were shown and C. percentage of cells
expressed EGFP was acquired through manual cell count. The data
shown were the mean.+-.s.e.m., n=4.
[0034] FIG. 21 shows micrographs images depicting that the
fluorescence of FITC-pDNA was quenched in differentiated, but not
native neuronal cells. A. Native and differentiated (10 .mu.M FSK)
NG-108 cells were transfected by LPEI/FITC-pDNA (N/P=20). Quenching
reagent, EtBr (20 .mu.g/ml), was added 4 h post transfection. Cell
images were taken before and after quenching by EtBr. Bar
represents 5 .mu.m. B. Cells were transfected by pre-complexed
LPEi/FITC- or Rhodamine-pDNA. Percentage of cells associated with
labelled pDNA was acquired after quenching of extracellular
fluorescence with EtBr or trypan blue, at 4 or 24 h post
transfection. Ratio of FITC/Rho in native and differentiated NG-108
cells was calculated. The data shown were the mean.+-.s.e.m.,
n=20.
[0035] FIG. 22 shows data depicting that the fluorescence of
FITC-pDNA was quenched in primary cortical neurons. A. Primary
cortical neurons were transfected by LPEI/FITC-pDNA (N/P=20).
Quenching reagent, EtBr (20 .mu.g/ml), was added 4 h post
transfection. Cell images were taken before and after quenching by
EtBr. Bar represents 5 .mu.m. B. Cells were transfected by
pre-complexed LPEI/FITC- or Rhodamine-pDNA. Percentage of cells
associated with labelled pDNA was acquired after quenching of
extracellular fluorescence with EtBr or trypan blue, at 4 or 24 h
post transfection. Ratio of FITC/Rho in primary cortical neurons
was calculated. The data shown were the mean.+-.s.e.m., n=20.
[0036] FIG. 23 shows histograms showing that polyplexes localized
differentially in native and differentiated neuronal cells.
LPEI/Rhodamine-pDNA (N/P=20) were used to transfect native or
differentiated (10 .mu.M FSK) NG-108 cells with centrifugation
transfection procedure. Four hours later, culture media was removed
and replenished with 1.times.PBS containing 50 nM Lysotracker green
DND-26 and the incubation continued for 5 min before observation by
confocal microscopy. Images of single cell were captured at
100.times. magnification. Co-localized pixels of Rhodamine with
lysotracker green DND-26 was analysed. The data shown were the
mean.+-.s.e.m., n=20.
[0037] FIG. 24 shows histograms visualising the amount of acidic
compartment increased after neuronal differentiation. Native and
differentiated (10 .mu.M RA) Neuro2A was incubated in PBS
containing Lysotracker Green. Total pixel of labelled acidic
compartment was obtained by summing pixels of Z-stack images (whole
cell). Significant differences in total pixels/cell were calculated
using the two tailed student's t-test. The data shown were the
mean.+-.s.e.m., n=20 for each phenotype. **, P<0.005.
[0038] FIG. 25 shows micrograph images, depicting that the
endosomal release of labeled pDNA happened after addition of
DOPE/CHEMS. Differentiated Neuro2A cells were transfected with
LEPI/Rhodamine-pDNA complexes (N/P=20), and further incubated for 4
h. Then, lysotracker green and DOPE/CHEMS were added and images
were taken continuously for 20 min. Real-time tracking demonstrated
release of polyplexes (indicated by asterisks) from the acidic
compartments. Measurement bar represents 5 .mu.m.
[0039] FIG. 26 shows histograms, depicting the effect of DOPE/CHEMS
being time-dependent. LPEI/pDNA (N/P=20) was used to transfect
differentiated (10 .mu.M RA) Neuro2A cells. DOPE/CHEMS was added at
different time point post transfection. Transfection efficiency was
acquired by counting fluorescent and bright field images for EGFP+
cells after 24 h of incubation. Percentage of EGFP positive cells
bearing neurites twice the cell body length was presented as
mean.+-.s.e.m (n=4). Significant differences in transfection
efficiencies were calculated using the two tailed student's t-test.
**, p<0.005.
[0040] FIG. 27 shows column graphs, the data of which indicates
that commercial reagents did not show cumulative enhancement of
transfection. Pre-complexed LPEI/pDNA (N/P=20) was used to
transfect differentiated (10 .mu.M RA) Neuro2A cells. Chloroquine
(25 .mu.M or 100 .mu.M), PLUS reagent (10 .mu.L or 20 .mu.L) or
INF7 fusogenic peptide (10 .mu.g or 20 .mu.g) was added
post-transfection. Transfection efficiency and cell viability was
acquired by counting fluorescent and bright field images for EGFP+
cells after 48 h of incubation. Percentage of EGFP positive cells
bearing neurites twice the cell body length was presented as
mean.+-.s.e.m (n=3). Significant differences in cell viability (%
of cell/image) against control (polyplexes only) were calculated
using the two tailed student's t-test. *, p<0.05, **,
p<0.005.
[0041] FIG. 28 shows column graphs, showing that LPEI/fusogenic
reagent-pDNA complexes did not yield a significant improvement of
transfection. Plasmid DNA (2 .mu.g) was incubated with PLUS reagent
(10 .mu.L or 20 .mu.L) or INF7 fusogenic peptide (10 .mu.g or 20
.mu.g) for 15 min prior to complexation with LPEI (N/P=20). The
polyplexes were then used to transfect differentiated (10 .mu.M RA)
Neuro2A cells. Transfection efficiency and cell viability was
acquired by counting fluorescent and bright field images for EGFP+
cells after 48 h of incubation. Percentage of EGFP positive cells
bearing neurites twice the cell body length was presented as
mean.+-.s.e.m (n=3). Significant differences in cell viability
against control were calculated using the two tailed student's
t-test. **, p<0.005.
[0042] FIG. 29 shows, as column graphs, that a minimal duration of
incubation with DOPE/CHEMS and Tubastatin A is required for high
transfection efficiency. LPEI/pDNA (N/P=20) were used to transfect
of differentiated Neuro2A (stimulated by 20 .mu.M RA). Cells were
treated with DOPE/CHEMS and Tubastatin A (5, 10 or 20 .mu.M) for 6,
8 or 12 h. Chemicals were removed by replacement with complete
media and cells were further incubated for 48 h. Transfection
efficiency was acquired by counting fluorescent and bright field
images for EGFP+ cells. Data presented group mean.+-.s.e.m (n=3).
Significant differences in transfection efficiencies were
calculated using the two tailed student's t-test. *, p<0.05.
[0043] FIG. 30 shows histograms depicting the data that DOPE/CHEMS
and Tubastatin A enhanced transfection efficiency was mediated by
some but not all cationic carriers. LPEI/pDNA (N/P=20), PAMAM/pDNA
(N/P=10), XtremeGene HP/pDNA (3 .mu.L: 1 .mu.g of pDNA) and Fugene
HD (1.5 .mu.L: 1 .mu.g of pDNA) were used to transfection
undifferentiated and differentiated Neuro2A (10 .mu.M RA). Cells
were treated with DOPE/CHEMS, Tubastatin A (10 .mu.M) or DOPE/CHEMS
and Tubastatin A (10 .mu.M) for 12 h. Control indicates cells
exposed to DNA complexes only. Chemicals were removed by
replacement with complete media and cells were further incubated
for 48 h. Transfection efficiency was acquired by counting
fluorescent and bright field images for EGFP+ cells. Data presented
group mean.+-.s.d. (n=3). Significant differences in transfection
efficiencies were calculated using the two tailed student's t-test.
*, p<0.05.
[0044] FIG. 31 shows a graph depicting the data that Trichostatin A
enhanced transfection. LPEI/pDNA (N/P=20) were used for
transfection of differentiated (10 .mu.M RA) Neuro2A cells. In the
presence of DOPE/CHEMS, Trichostatin A (50 and 100 nM) was added 1
h post transfection. Transfection efficiency was acquired 24 h
later by counting fluorescent and bright field images for EGFP+
cells after 24 h of incubation. Percentage of EGFP positive cells
bearing neurites twice the cell body length was presented as
mean.+-.s.e.m (n=3).
[0045] FIG. 32 shows histograms representing the data showing that
DOPE/CHEMS and Tubastatin A led to high transfection efficiency and
low toxicity in primary cortical neurons. LPEI/pDNA (N/P=20) were
used to transfect of primary cortical neurons by centrifugation
transfection procedure. Cells were incubated in neurobasal media
containing DOPE/CHEMS and Tubastatin A (16 .mu.M) for 24 h.
Chemicals were removed by replacement with fresh neurobasal media
and cells were further incubated for 24 h. A. Cell viability was
obtained by counting total number of cells per bright field image
and normalized by control (without DOPE/CHEMS and Tubastatin A).
Data presented group mean.+-.s.e.m (n=4). B. In the presence of
DOPE/CHEMS, Tubastatin A (16 .mu.M) was added to the primary
cortical neurons culture immediately, 1, 2 and 3 h post
transfections. Transfection efficiency was quantified by FACS
analysis 48 h later and presented as mean.+-.s.d (n=3). Significant
differences in transfection efficiencies were calculated using the
two tailed student's t-test. *, p<0.05; **, p<0.005.
[0046] FIG. 33 shows micrographs of HDAC6 inhibition and that
treatment with paclitaxel resulted in tubulin acetylation.
Differentiated Neuro2A cells (pre-treated with 10 .mu.M RA) were
exposed to HDAC inhibitors such as Tubastatin A (10 .mu.M), TSA (50
nM), Paclitaxel (25 nM), SAHA (5 .mu.M), Entinostat (10 .mu.M), or
Tacedinaline (50 .mu.M) for 2 h. Then, cells were fixed with 4%
formaldehyde and co-stained for acetylated .alpha.-tubulin and
.beta.-tubulin. Confocal images of the individual and merged
channels are shown. Bar represents 20 .mu.m. Further, a table is
presented, showing the reagents and their respective targets, as
well as the concentration at which the experiment was done.
[0047] FIG. 34 presents data showing that treatment with DOPE/CHEMS
and HDAC6 targeting inhibitors resulted in unprecedented
transfection efficiency. LPEI/pDNA (N/P=20) were used to transfect
of differentiated neuronal cells (10 .mu.M RA) by centrifugation
transfection procedure. Then, cells were exposed to DOPE/CHEMS and
Tubastatin A (10 .mu.M), TSA (50 nM), Paclitaxel (25 nM), SAHA (5
.mu.M), Entinostat (10 .mu.M), or Tacedinaline (50 .mu.M) for 12 h.
Cell exposed to DOPE/CHEMS served as control. Transfection
efficiency was quantified by FACS analysis 48 h later and presented
as mean.+-.s.d (n=3). Significant differences in transfection
efficiencies were calculated using the two tailed student's t-test.
*, p<0.05; **, p<0.005.
[0048] FIG. 35 depicts micrographs showing the transient effect of
Tubastatin A on tubulin acetylation. Neuro2a cells were treated
with Tubastatin A (10 .mu.M) for 12 hours. Then, chemicals were
removed by replacement with complete media and cells were further
incubated for 48 h. At various time points, cells were fixed with
4% formaldehyde and stained for acetylated .alpha.-tubulin (Green)
and nucleus (Hoechst stain, Blue). Representative images were
shown. Bar represents 20 .mu.m.
[0049] FIG. 36 shows data indicating that neurite outgrowth was not
affected by LPEI mediated transfection. Neuro2A cells were
transfected by LPEI-pDNA (N/P=20) in the presence or absence of
DOPE/CHEMS and Tubastatin A (10 .mu.M). Twelve hour post
transfection, cell culture media was removed and replaced with DMEM
containing 1% FBS. Six hours later, neuronal differentiation was
stimulated by 10 .mu.M RA/1% FBS/DMEM. After 48 h of incubation,
cells were fixed with 4% formaldehyde and stained for nucleus
(Hoechst stain, Blue) and imperial protein stain. A. shows
micrographs of representative images. Bar represents 50 .mu.m. B.
shows a histogram showing the average neurite length, which was
measured by HCA vision and presented as mean of pixel.+-.s.e.m
(n=80-100, biological quadruplicates).
[0050] FIG. 37 shows various line graphs, depicting the recovery of
global metabolism after washout of DOPE/CHEMS and Tubastatin A.
Neuro2A cells were transfected by LPEI-pDNA (N/P=20) in the
presence or absence of DOPE/CHEMS and Tubastatin A (10 .mu.M).
Negative control indicates cells without treatment throughout the
experiment. Twelve hour post-transfection, chemicals were removed
by replacement with fresh complete media. Cells were then collected
at 4, 24, and 48 h later and subjected to LC/MS analysis for
various pathways including A. TCA cycle, B. Glycolysis, C. Glycogen
metabolism, D. Nucleotide metabolism, E. Phospholipid synthesis and
F. Tryptophan metabolism. At each time point, quantriplicates of
negative control (C), cells exposed to polyplexes (P) and cells
exposed to both polyplexes, DOPE/CHEMS and Tubastatin A (T) were
collected. Relative fold change to negative control was obtained
after normalization of each metabolite to the ATP readout,
presented as mean.+-.s.d. (n=4). Significant differences in the
relative fold change were calculated using the two tailed student's
t-test. *, p<0.05.
[0051] FIG. 38 shows a histogram visualising the data pertaining to
polyplex sedimentation. LPEI/pDNA (various N/P ratios) was
incubated in DMEM for 15 min or 4 h. At the end of incubation, some
samples were centrifuged and all were subsequently treated with
pAA/urea lysis buffer. The amount of pDNA in the supernatant was
measured by qPCR (normalized to control where N/P=0). The data
shown were the mean.+-.s.e.m., n=3.
[0052] FIG. 39 shows a schematic of the experiment design used in
testing the contribution of aggregated polyplexes to transfection.
LPEI/pDNA (N/P=20) pre-complexed in DMEM (with or without
filtration through 0.22 .mu.m) was centrifuged in the presence or
absence of Neuro2a cells. Transfection mixture was replaced by
complete media and incubated for 48 h. Transfection efficiency was
quantified by flow cytometry.
[0053] FIG. 40 shows column graphs depicting that aggregated
polyplexes mediated efficient transfection. A. Neuro2A cells were
transfected with LPEI/pDNA (N/P=20) pre-complexed in DMEM (with or
without filtration) by bolus or deposited mediated transfection
procedures. Transfection efficiency was quantified by FACS 48 h
later. The data shown are the mean.+-.s.d., n=3. B. Neuro2A cells
were transfected by LPEI complexed with various amount of pDNA at
N/P=0, 10, 20 via deposit mediated transfection procedure.
Transfection efficiency was quantified by FACS after 48 h
incubation. The data shown were the mean.+-.s.e.m., n=3.
[0054] FIG. 41 shows a data that aggregated polyplexes were removed
by 0.22 .mu.M filter. LPEI/pDNA (N/P=20) pre-complexed in HEPES was
filtered. After which, concentrated DMEM (18.times.) was added to
the filtrate to a final concentration of 1.times.DMEM and incubated
for 15 min. The mixture (filtered or non-filtered) was then added
to plated Neuro2A cells and centrifuged. Cells were not transfected
using filtrate (insert). Transfection efficiency was quantified by
FACS 48 h later. The data shown were the mean.+-.s.d., n=4.
Furthermore, a schematic of the experimental design done is
shown.
[0055] FIG. 42 shows data, in form of a histogram, indicating that
LPEI/pDNA aggregated extensively DMEM. LPEI/pDNA (N/P=20) in 25 mM
HEPES or DMEM and incubated for 15 min. Absolute copy number of
pDNA, with or without filtration (HEPES or DMEM), and in HEPES that
was added to 18.times.DMEM, with or without second step of
filtration (HEPES+18.times.DMEM) were quantified by qPCR after
treatment with pAA/urea lysis buffer. The data shown were the
mean.+-.s.e.m., n=3.
[0056] FIG. 43 shows a histogram, whereby the data indicates that
the pDNA had not efficiently released from the surface bound
aggregate. In the presence or absence of Neuro2A, transfection
mixture of LPEI/pDNA (N/P=20) was centrifuged and replenished with
complete media. At various time points, cells were harvested by
trypsinization and supernatant collected. Samples were then treated
with pAA/urea lysis buffer. The absolute copy number of pDNA was
quantified by qPCR and normalized by control, N/P=0. The data shown
were the mean.+-.s.e.m., n=3.
[0057] FIG. 44 demonstrates the effect of TrafEn.TM. in enhancing
polymer-based transfection. This was tested on different cells and
cell types, for example human glioma/breast cancer cell lines,
human mesenchymal stem cells (hMSCs), murine Abelson leukemia virus
induced tumour cells (RAW 264.7), human prostate cancer cells
(PC3), human melanoma cells (M14) and mouse embryonic fibroblast
cells (MEFs). Cells were transfected with PEIMAX/2 .mu.g PMAXGFP
(LONZA). After transfection, cells were incubated in culture media
in the presence or absence of the TrafEn.TM. reagent. Forty-eight
hours later, GFP+ cells were analysed by FACS analysis or
semi-automated cell count using Image J. The histograms present
percentage of GFP+ cells and error bar represent S.E.M of the
biological triplicate and technical duplicates. Representative
images are presented (Blue, Nucleus; Green, GFP).
[0058] FIG. 45 shows the effect of TrafEn.TM. in the enhancement of
polymer-based transfection. MEF and M14 cells were transfected with
EV71-pIRES-eGFP expression vector complexed with various
transfection reagents. Transfections were conducted using 2
protocols--MI and IP. MI refers to the manufacture's instruction.
IP refers to an in-house protocol, which is the deposit mediated
transfection described in the specification, for example in FIG.
39. IP+ TrafEn.TM. refers to the addition of TrafEn.TM. reagents in
the culture media after transfection. The histograms here represent
the percentage (%) of GFP+ cells for each procedure, each using a
different transfection reagent.
[0059] FIG. 46 shows histograms depicting the efficient knockdown
of PTB1. HeLa cells were transfected with Scramble 30012 (Control),
and PTB shRNA namely TR302218A 8865 (1), TR302218B 8866 (2),
TR302218C 8867 (3), or TR302218D 8868 (4). The shRNAs were
delivered by PEIMAX (N/P=10) in the presence or absence of
TrafEn.TM. (DOPE/CHEMS+10 .mu.M Tubastatin A). Three days post
treatment cells were harvested for qPCR analysis. Graph present
fold change of PTB1 to control after normalization to GAPDH.
[0060] FIG. 47 shows micrographs depicting the efficient knockdown
of PTB, resulting in rapid transdifferentiation. HeLa cells were
transfected with Scramble 30012 (Control), PMAXGFP (Control), and
TR302218C 8867. The shRNAs were delivered by PEIMAX (N/P=10) in the
presence or absence of TrafEn.TM. (DOPE/CHEMS+10 .mu.M Tubastatin
A). Post transfection, cells were treated with Puromycin and
culture in N3 media (Media for neuronal cells). Images were
captured up to 8 days. Then, cells were fixed with 4% Formaldehyde
and stained with Tuj antibody.
[0061] FIG. 48 shows graphs representing percentage (%) of cells
showing low/high expression and the total % of GFP+ cells.
Furthermore, representative images of the FACS analysis and
transfected cells for each conditions are presented. Error bar
represents the standard error mean (S.E.M.) of n=3. Statistical
significance of transfection efficiencies between cells treated
with DOPE/CHEMS+SAHA and DOPE/CHEMS or SAHA were obtained using two
tailed student's t-test; *, p<0.01 the combinatorial effect of
DOPE/CHEMS and SAHA or Tubastatin A in enhancing transfection.
U251MG cells were transfected with PEIMAX complexed with 1.5 .mu.g
of PMAXGFP at N/P=10 in the absence or presence of DOPE/CHEMS
and/or HDACi (10 .mu.M Tubastatin A, 5 .mu.M SAHA). After 24 h of
transfection, culture media were replaced with fresh media with or
without SAHA/Tubastatin A and further incubated for 48 h. Next,
cells were harvested and GFP expressions were analysed by FACS
analysis.
[0062] FIG. 49 shows fluorescent micrographs and histograms showing
the synergistic effect of p53 and transfection enhancers. Cells
were transfected with 1.5 .mu.g of PMAXGFP or pEGFP-N1-p53 (p53).
Transfection enhancers (DOPE/CHEMS [D] and 5 .mu.M SAHA) were added
to the culture media post transfection. After 24 h of transfection,
culture media were replaced with fresh media with or without SAHA
and further incubated for 48 h. Then, cells were fixed with 4%
Formaldehyde and stained with Hoechst 33342. Fluorescent images
were captured and nucleus number was counted with Image J.
Percentage of viable cells represents % of cell to negative control
(-ye). The lower panel contains representative images from each
condition. Error bar represents the standard error mean (S.E.M.) of
n=3. Unpaired, student t-test was performed to examine statistical
significance between cells treated with P53, D and SAHA to cells
treated with either one reagent. **, p<0.001.
[0063] FIG. 50 shows a graph depicting the percentage (%) of viable
cells, as well as fluorescent images of these cells, after
prolonged incubation of U251MG cells with SAHA, showing that this
did not increase cell death. Cells were transfected with
pEGFP-N1-p53 (p53). Transfection enhancers (DOPE/CHEMS and 5 .mu.M
SAHA) were added to the culture media post transfection. The cells
were further treated with SAHA for 48, 72 and 96 h. Next, cells
were fixed with 4% Formaldehyde and stained with Hoechst 33342.
Fluorescent images were captured and nucleus number was counted
with Image J. Percentage of viable cells represents % of cell over
cells transfected with p53 only. The lower panel contains
representative images from each condition. Unpaired, student t-test
was performed to examine statistical significance between cells
treated with SAHA for 48 h and 72 or 96 h. **, p<0.001.
[0064] FIG. 51 shows a graph and accompanying fluorescent images,
showing the synergistic cytotoxic effect of p53 with SAHA, but not
Tubastatin A. Cells were transfected with PMAXGFP or pEGFP-N1-p53
(p53). Transfection enhancers (DOPE/CHEMS [D], 10 .mu.M Tubastatin
A [T] and 5 .mu.M SAHA) were added to the culture media
individually or combination post transfection. Culture media were
replaced with fresh media with or without Tubastatin A or SAHA and
further incubated for 48 h. Then, cells were fixed with 4%
Formaldehyde and stained with Hoechst 33342. Fluorescent images
were captured and nucleus number was counted with Image J.
Percentage of viable cells represents % of cell to cells
transfected with PMAXGFP. The lower panel consists of
representative images from each condition. Error bar represents the
standard error mean (S.E.M.) of n=3. Unpaired, student t-test was
performed to examine statistical significance between 2 treatment
conditions. **, p<0.001.
[0065] FIG. 52 shows fluorescent micrographs and histograms
depicting the synergistic effect of p53 with SAHA in inducing
significant cell death in both TMZR-U251MG and GSC. (A) TMZR-U251MG
and (B) GSC cells were transfected with PMAXGFP and pEGFP-N1-p53
(p53) in the presence of DOPE/CHEMS [D] and SAHA individually or in
combination. Twenty-two hours post transfection, the cell media was
replaced with DMEM/10% FBS/40 .mu.M TMZ for TMZR-U251MG and
DMEM/serum replacement for GSC, with or without SAHA. Cells were
further incubated for 48 h. Cells were fixed with 4% Formaldehyde
and stained with Hoechst 33342. The images were analysed by Image
J. Representative images are presented. Graphs present average of %
of cells over control cells exposed to DOPE/CHEMS only (negative
control+D). Error bar represents the standard error mean (S.E.M.)
of n=3. Unpaired, student t-test was performed to examine
statistical significance between control and p53 transfected cells.
**, p<0.001.
[0066] FIG. 53 shows fluorescent micrographs and histograms
visualising the effect of DOPE/CHEMS and Trichostatin A (TSA) on
the enhancement of transfection. The human fetal MSC cells were
transfected with PEIMAX complexed with 1.5 .mu.g of PMAXGFP at
N/P=10 in the absence or presence of DOPE/CHEMS and/or HDACi (100
nM Trichostatin A). After 24 h of transfection, culture media were
replaced with fresh media with or without Trichostatin A and
further incubated for 48 h. Next, cells were harvested and GFP
expressions were analysed by FACS analysis. Graph represents % of
GFP+ cells. Error bar represents the standard error mean (S.E.M.)
of n=3. Statistical significance of transfection efficiencies
between cells exposed to polyplexes only and other conditions were
obtained using two tailed student's t-test; *, p<0.01.
Representative images of the transfected cells of each condition
are presented.
[0067] FIG. 54 depicts histograms depicting expression levels of
BMP2 in human MSC under various treatment conditions. Human fetal
MSC cells were transfected with PEIMAX complexed PMAXGFP (control)
or PMAXGFP-BMP2 (human BMP2 cloned into PMAXGPF vector) at N/P=10
(BMP2). Post transfection, cells were treated with or without
DOPE/CHEMS, 5 .mu.M Tubastatin A, and 150 nM TSA in combination or
individually. 24 hours post transfection, culture media was
replaced with fresh media containing HDACi according. After 48 h of
incubation, cells were trypsinized and total RNA were collected
with RNeasy Mini Kit (Qiagen) to avoid DNA contamination. One
microgram (.mu.g) of the total RNA was reversed transcribed and the
expression of BMP2 was measured using qPCR. The threshold cycles
(C.sub.ts) of BMP2 were normalized to the house-keeping gene GAPDH.
Cells transfected with PEIMAX/PMAXGFP serves as a negative control
(control). Graph presents fold change of BMP2 expression to the
negative control. Error bar represents S.E.M of means (n=3).
Unpaired, student t-test was used to test statistical significance
between treatment conditions. *, p<0.01.
[0068] FIG. 55 shows bright field and fluorescent images of GFP
Expression of in human MSC up to 21 days of incubation. Human fetal
MSC cells were transfected with PEIMAX/PMAXGFP complex at N/P=10.
Post transfection, the media was replaced with fresh culture media
with or without DOPE/CHEMS and 150 nM TSA. Three days after
transfection, media was replaced with fresh culture media. Bright
field and fluorescent images were taken at Day 2, 7, 14 and 21 days
post transfection. Representative imaged are presented.
[0069] FIG. 56 shows bright field images of MSC cells from 3 up to
21 days post transfection. MSC cells were transfected with PEIMAX
mediated delivery of PMAXGFP or PMAXGFP-BMP2. After transfection,
cells were treated with DOPE/CHEMS and 150 nM TSA individually or
in combination. 72 hours post transfection, culture media was
replaced with (A) expansion media alpha-MEM/10% FBS or (B)
osteogenic differentiation media (alpha-MEM supplemented with 10%
FBS, 10 mM .beta.-glycerophosphate, 10 nM dexamethasone, and 0.2 mM
ascorbic acid. Cells were then further incubated and the respective
media was replaced every 3 days.
[0070] FIG. 57 shows micrograph images (bright field and
fluorescent), as well as column graphs and FACS analysis graphs
visualising the combinatorial effect of DOPE/CHEMS and SAHA or
Tubastatin A in the enhancement of transfection. The PC3 cells were
transfected with PEIMAX complexed with 1.5 .mu.g of PMAXGFP at
N/P=20 in the absence or presence of DOPE/CHEMS [D] and/or HDACi
(20 .mu.M Tubastatin A [T], 5 .mu.M SAHA). After 24 h of
transfection, culture media was replaced with fresh media with or
without SAHA/Tubastatin A and further incubated for 48 h. Next,
cells were harvested and GFP expression was analysed by FACS
analysis. The column graph shows the percentage (%) of cells
considered to be showing low/high expression of GFP and the total %
of GFP+ cells. Representative images of the FACS analysis and
transfected cells of each condition are presented. Error bar
represents the standard error mean (S.E.M.) of n=3.
[0071] FIG. 58 shows histograms depicting the expression levels of
GM-CSF in PC3 cells under various treatment conditions. Human PC3
cells were transfected with PEIMAX complexed PMAXGFP (control) or
PMAXGFP-GM-CSF (human GM-CSF cloned into PMAXGPF vector, GMCSF) at
N/P=20. Post transfection, cells were treated with or without
DOPE/CHEMS [D] and 5 .mu.m SAHA individually or in combination
[TrafEn.TM.]. 24 hours post transfection, culture media was
replaced with fresh media containing HDACi accordingly. After 48 h
of incubation, cells were trypsinized and the total RNA was
collected individually using the RNeasy Mini Kit (Qiagen) to avoid
DNA contamination. One microgram (.mu.g) of total RNA was
reverse-transcribed and the expression of GM-CSF was measured using
qPCR. The threshold cycles (C.sub.ts) of GM-CSF were normalized to
the house-keeping gene GAPDH. Untransfected cells served as
controls (Ctrl).
[0072] FIG. 59 shows histograms visualising that SAHA induced an
up-regulation of MICA and NKG2D in PC3 cells. Human PC3 (prostate
cancer) cells were treated with 5 .mu.M SAHA for 8, 20 and 48 hours
(hr). At the end of the incubation, the cells were collected and
subjected to PCR analysis. Cells with no exposure to SAHA (Negative
8 hr) served as a control. The histogram here represented fold
change to control after normalization with GAPDH.
[0073] FIG. 60 show a high N/P ratio for transfection of HaF. Cells
were transfected by PEIMAX/2 .mu.g PMAXGFP at various N/P ratio
using deposit mediated transfection protocol. Forty eight hour
later, cells were trypsinized and analysed by flow cytometry. The
percentage of cells expressed GFP is presented and error represents
S.D. for n=3.
[0074] FIG. 61 show data showing that a high transfection resulted
in efficient reprogramming of HaF. (A) Fibroblast cells were
transfected with PEIMAX (N/P=50), Lipofectamine or Satisfection
complexed to 2 .mu.g PMAXGFP. After 48 h incubation, cells were
trypsinized and analysed by flow cytometry analysis. Graph presents
average % of GFP+ cells (n=3). Fluorescent images of the GFP+ cells
are presented. (B, C) Fibroblast cells were transfected with PEIMAX
complexed to 2 .mu.g PMAXGFP or polycistronic OSKM (Addgene: 20328)
at N/P=50. Next, the transfection mixture was replaced with culture
media with or without TrafEn.TM. (DOPE/CHEMS+10 .mu.M Tubastatin
A). Two days later, the media were replaced with fresh culture
media (10% FBS/DMEM). After 24 h incubation, total RNA were
isolated with Qiagen RNAeasy kit and subjected to qPCR analysis.
Graph presents expression of OSKM to control (cells transfected
with PMAXGFP) after normalization to GAPDH. For a second set of
experiment, cells were further incubated for 7 days. At the end of
experiment, cells were fixed with 4% formaldehyde and stained with
Oct4 antibody.
[0075] FIG. 62 shows histograms showing that VPA induced
up-regulation of KLF4 and c-Myc in HaF. Fibroblast cells were
treated with 1 mM VPA for up to 3 days. At the end of incubation,
the cells were collected and subjected to PCR analysis. Cells with
no exposure to VPA (3 days) serve as control. Graphs present fold
change to control after normalization with GAPDH.
DEFINITION OF TERMS
[0076] As used herein, the term "nucleic acid" designates a
molecule comprising one or more nucleotides, or an oligonucleotide,
or a fragment thereof, including, without limitation, ribonucleic
acid (RNA), messenger RNA (mRNA), DNA/RNA hybrids, non-natural or
synthetic nucleic acids, short interfering RNA (siRNA), short
hairpin RNA (shRNA), deoxyribonucleic acid (DNA), plasmid DNA
(pDNA), antisense and sense oligonucleotides, nucleotides or
combinations thereof. The nucleic acid may be single-stranded, or
partially or completely double-stranded (duplex). Duplex nucleic
acids may be homoduplex or heteroduplex. The term "ribonucleic
acid" (RNA) refers to biomolecules that play an important role in
the regulation, coding, decoding and expression of genes. Each
ribonucleic acid consists of a nucleotide, either adenine (A),
cytosine (C), guanine (G) or uracil (U), and a ribose sugar. A
ribonucleic acid sequence comprises of a chain of these nucleic
acids, resulting in a sugar-phosphate backbone. Concurrently, for
the term "deoxyribonucleic acid" (DNA) refers to a biomolecule,
consisting of either adenine (A), cytosine (C), guanine (G) or
thymidine (T), attached to the sugar/phosphate to form the complete
nucleotide.
[0077] As used herein, the term "isolated" means that a nucleotide
sequence, for example a gene, primer, or oligonucleotide or other
sequence is substantially or essentially free from other nucleic
acids or other impurities.
[0078] As used herein, the term "amplicon", "amplified product" or
"amplification product" refers to a product of an amplification
reaction. An example of an amplicon is a nucleotide sequence
produced as a result of PCR, real-time PCR, reverse
transcription-PCR, competitive RT-PCR, ligase chain reaction (LCR),
gap LCR, strand displacement amplification (SDA), nucleic acid
sequence based amplification (NASBA), transcription-mediated
amplification (TMA), rolling circle amplification (RCA) or the
like.
[0079] The term "primer" is used herein to mean any single-stranded
oligonucleotide sequence capable of being used as a primer in, for
example, PCR or RCA technology. Thus, a "primer" according to the
disclosure refers to a single-stranded oligonucleotide sequence
that is capable of acting as a point of initiation for synthesis of
a primer extension product that is substantially identical to the
nucleic acid strand to be copied (for a forward primer) or
substantially the reverse complement of the nucleic acid strand to
be copied (for a reverse primer). A primer may be suitable for use
in, for example, PCR technology. Single-stranded includes, for
example, hairpin structures formed by single-stranded nucleotide
sequences. The design of a primer, for example its length and
specific sequence, depends on the nature of the target nucleotide
sequence and on the conditions at which the primer is used, for
example, temperature and ionic strength.
[0080] The primers may consist of the nucleotide sequences
described herein, or may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,
100 or more nucleotides which comprise or fall within the sequences
described herein, provided they are suitable for specifically
binding a target nucleic acid sequence, under stringent conditions.
In one embodiment, the primer sequence is less than 35 nucleotides
in length, for example the primer sequence is less than 34, 33, 32,
31, 30, 29, 28, 27, 26, 25, 24, 23, 22 21 20 19 18 17 16 15 14 13
12 11 or 10 nucleotides in length. Slight modifications of the
primers or probes, in length or in sequence, can be carried out to
maintain the specificity and sensitivity required under the given
circumstances. In one embodiment of the present disclosure, probes
and/or primers described herein may be extended in length by 1, 2,
3, 4 or 5 nucleotides or reduced in length by 1, 2, 3, 4 or 5
nucleotides, for example, in either direction. Primer sequences can
be synthesized using any methods well known in the art.
[0081] As used herein, the terms "amplification" refers to an
amplification reaction, for example an enzyme-mediated reaction
used to amplify a specific target nucleotide sequence. By
amplifying the target nucleotide sequence, the reaction produces
many more copies of the target nucleotide sequence to produce an
amplicon, amplified product or amplification product. One example
of an amplification reaction is a "polymerase chain reaction'
(PCR)". PCR is carried out with the aid of thermal cycler in a
mixture containing a polymerase enzyme, a set of primers, for
example a set of forward and reverse primers and any additional
primers that may be required and four deoxynucleotide
triphosphates--dNTPs).
[0082] As used herein, the terms "therapeutic gene" and gene
therapy refer to a therapy for genetic disorders, often similar to
therapy for other disorders. Gene therapy may involve insertion of
normal copies of a gene into the cells of people that is in vivo,
with a specific genetic disorder. This therapy may involve
replacing a deficient compound or blocking an overactive pathway.
Gene therapy may also involve turning off genes. Genetic
modification may also be used in ex vivo gene therapy. For example,
human stem cells, immune cells or cancer cells, can be genetically
modified for various applications. Cells are modified to induce
differentiation, transdifferentiation or reprogramming. Also, cells
may be modified to serve as vehicle to deliver therapeutic protein.
For example, mesenchymal stem cells may be modified to overexpress
BMP2.
[0083] The term "polyplex" refers to complexes of genetic material
and a cationic species. The ratio of genetic material to cationic
species (nucleic acid:polymer (N/P)) may be selected from the group
consisting of from about 0 to about 1000, about 0 to about 900,
about 0 to about 800, about 0 to about 700, about 0 to about 600,
about 0 to about 500, about 0 to about 400, about 0 to about 300,
about 0 to about 200, about 0 to about 100, about 0 to about 75,
about 0 to about 50, about 0 to about 25, about 0 to about 20,
about 0 to about 15, about 0 to about 10, and about 0 to about
5.
[0084] The term "anti-cancer drugs", also known as chemotherapeutic
agents, refers to agents which used to treat cancers of the human
body. There are different kinds of chemotherapeutics, which are
defined into groups based on their method of action. Examples of
the different groups of chemotherapeutics known in the art are as
follows: alkylating antineoplastic agents, anti-metabolites,
anti-microtubule agents, topoisomerase inhibitors and cytotoxic
antibiotics. The agents can be administered to the patient
intravenously, orally or intrathecally. Isolated limb perfusion is
also a known delivery method for chemotherapeutics in certain
cases.
[0085] The term "taxanes" refers to a class of are diterpenes
produced by the plants of the genus Taxus (yews). Taxane based
chemotherapeutic regimes are widely prescribed for cancer patients.
Taxanes, which comprise of paclitaxel and docetaxel, promote
microtubule stabilization, and disrupt transition from metaphase to
anaphase. This blocks progression of cell division and prolonged
activation of the mitotic checkpoint induces apoptosis or reversion
to the G-phase, eventually causes cell death. Taxanes may be
selected from a group consisting of cremophor EL.RTM.
Taxoprexin.RTM.(Docosahexaenoic acid-paclitaxel), Xytotax.TM.
(paclitaxel polyglumex), TOCOSOL.RTM. paclitaxel, BMS-184476,
DJ-927, BMS-275183, RPR 109881A, Ortataxel, Genexol (co-polymer
combination), LEP (liposomal-encapsulated paclitaxel) and taxol in
vitamin E emulsion.
[0086] The term "epothilone" as used herein represents an emerging
class of drugs for cancer treatment. The mechanism of action for
epothilone class is similar to taxanes, which is the blockage of
mitosis and induction of apoptosis. Nevertheless, Epithilones were
shown to be more potent and milder side effects than taxanes.
Additionally, their better water solubility characteristic enables
the replacement of cremophors (solubilizing agents of paclitaxel)
which was shown to affect cardiac function.
[0087] The term "histone deacetylase inhibitor" (HDACi) refers to a
class of compounds that interfere with the function of histone
deacetylase. HDACi is also known as epigenetic modifier. For
example, by modifying the epigenetic pattern, SAHA and TSA have
shown to exhibit various activities, such as immunomodulation and
apoptosis. Although the clinical use of HDACi is widely associated
with anti-cancer treatment, HDACi has also been investigated as
therapeutic intervention for neurodegenerative disease, an
anti-inflammatory and for the protection of heart muscle. More
recently, HDACi has been demonstrated to promote self-renewal and
enhance differentiation of stem cells, as well as increasing
reprogramming efficiency of somatic cells. HDACs are also known to
be involved in the maintenance and function of chromatin via
regulation of acetylation state of histone. Advantageously, given
such global effects on histone modulation, HDACi influences a broad
repertoire of physiological processes, including transcription of
genes involved in proliferation, differentiation, survival and DNA
repair.
[0088] The term "TrafEn.TM." stands for trafficking enhancer and
relates to two agents directing the genetic material or complex
containing genetic material to a productive pathway for efficient
transfection. In particular, TrafEn.TM. relates to transfecting a
cell using comprising a first agent capable of directing the
genetic material away from the acidic compartments and a second
agent capable of stabilizing the microtubule or a network thereof.
The application of TrafEn.TM. may be extended further to
chemosensitize cells by rationally designing the composition to
achieve a specific therapeutic effect. The first agent, as defined
above, may also be termed "chemoRe-router".
[0089] A "subject" or an "individual" is a living multi-cellular
vertebrate organism. In the context of this disclosure, the subject
can be an experimental subject, such as a non-human animal, e.g., a
mouse, a cotton rat, or a non-human primate. Alternatively, the
subject can be a human subject.
[0090] The terms "biological material" or "biological sample" as
used herein refers to any material or sample, which includes an
analyte as defined herein. Such samples may, for example, include
samples derived from or comprising stool, whole blood, serum,
plasma, bone marrow, tears, saliva, nasal fluid, sputum, ear fluid,
genital fluid, breast fluid, milk, colostrum, placental fluid,
amniotic fluid, perspiration, synovial fluid, ascites fluid,
cerebrospinal fluid, bile, gastric fluid, aqueous humor, vitreous
humor, gastrointestinal fluid, exudate, transudate, pleural fluid,
pericardial fluid, semen, upper airway fluid, peritoneal fluid,
fluid harvested from a site of an immune response, fluid harvested
from a pooled collection site, bronchial lavage, urine, biopsy
material, e.g. from all suitable organs, e.g. the lung, the muscle,
brain, liver, skin, pancreas, stomach, etc., a nucleated cell
sample, a fluid associated with a mucosal surface, hair, or
skin.
[0091] The terms "treatment", "therapeutic intervention" and
"therapy" may be used interchangeably herein (unless the context
indicates otherwise) and these terms refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
aim is to try and prevent or slow down (lessen) the targeted
pathologic condition or disorder. In tumor treatment, the treatment
may directly decrease the pathology of tumor cells, or render the
tumor cells more susceptible to treatment by other therapeutic
agents, e.g., radiation and/or chemotherapy. The aim or result of
tumor treatment may include, for example, one or more of the
following: (1) inhibition (i.e., reduction, slowing down or
complete stopping) of tumor growth; (2) reduction or elimination of
symptoms or tumor cells; (3) reduction in tumor size; (4)
inhibition of tumor cell infiltration into adjacent peripheral
organs and/or tissues; (5) inhibition of metastasis; (6)
enhancement of anti-tumor immune response, which may, but does not
have to, result in tumor regression or rejection; (7) increased
survival time; and (8) decreased mortality at a given point of time
following treatment. Treatment may entail treatment with a single
agent or with a combination (more than two) of agents. An "agent"
is used herein broadly to refer to, for example, a drug/compound or
other means for treatment e.g. radiation treatment or surgery.
Examples of treatment include surgical intervention, liver
transplantation, immunotherapy, chemotherapy with a given drug or
drug combination, radiation therapy, neo-adjuvant treatment, diet,
vitamin therapy, hormone therapies, gene therapy, cell therapy,
antibody therapy etc. The term "treatment" also includes
experimental treatment e.g. during drug screening or clinical
trials.
[0092] Additionally, the terms and expressions employed herein have
been used as terms of description and not of limitation, and there
is no intention in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the inventions embodied
therein herein disclosed may be resorted to by those skilled in the
art, and that such modifications and variations are considered to
be within the scope of this invention.
[0093] The invention has been described broadly and generically
herein. Each of the narrower species and sub-generic groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0094] Other embodiments are within the following claims and
non-limiting examples. In addition, where features or aspects of
the invention are described in terms of Markush groups, those
skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0095] Before the present inventions are described, it is to be
understood that this invention is not limited to particular
embodiments described, as such may vary. It is also to be
understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only in the appended claims.
[0096] Attaining high transfection efficiencies when delivering
genetic material, e.g. plasmid DNA and shRNA offers the potential
for the treatment of a myriad of devastating disorders including,
but not limited to, cancers, neurodegenerative diseases and
inflammatory disease, for which there are currently few treatment
options. Presently, low transfection and delivery efficiencies
limit the application of drug-gene therapeutics. The development of
galenics and methods for using this technology to enhance gene
delivery ex vivo and in vivo represent an unmet need in this
industry.
[0097] Advantageously, the present disclosure provides a unique
composition of biocompatible reagents (TrafEn.TM.), designed to
drastically enhance the gene delivery and simultaneously
chemosensitize the many types of hard-to-infect cells. The
drug-gene combination described herein relates to a strategy,
whereby a class of microtubule targeting chemosensitizers and a
chemoRe-router increase the delivery of a therapeutic gene. The
synergistic effect of TrafEn.TM. and the therapeutic gene are
thought to result in a superior therapeutic effect. The formulation
of chemosensitizers, as described further in the examples below,
may contain an optimized mix of fusogenic molecules that
specifically redirect carrier/DNA complexes away from the
non-productive acidic compartment, re-rerouting them onto then
microtubular networks stabilized by the use of
chemosensitizers.
[0098] Accordingly, the present disclosure provides a composition
for transfecting a cell with a genetic material, comprising a first
agent capable of directing the genetic material away from an acidic
compartment in a cell and a second agent capable of stabilizing the
microtubule or a network thereof.
[0099] In one embodiment, the first agent may be capable of
directing genetic material away from a non-productive acidic
compartment of the cell. In another embodiment, the first agent may
be, but is not limited to, a lipid, a peptide fusiongenic agent or
a combination thereof.
[0100] In one embodiment, the lipid fusogenic agent may be selected
from DOPE, CHEMS, DPPC and DOPC and combinations thereof.
[0101] In one embodiment, the peptide fusogenic agent may be at
least any one of, but not limited to, haemagglutinin (HA2-peptide),
influenza-derived fusogenic peptide diINF-7, T domain of Diphtheria
toxin and polycationic peptides, such as polylysine and
polyarginine, or combinations thereof.
[0102] In one embodiment, the aforementioned peptide fusogenic
agent may be chemically modified by attachment of a lipid.
[0103] In one embodiment, the aforementioned peptide may be
chemically modified by attachment of a biomolecule, such as a
nucleic acid or a synthetic carrier, such as a cationic
polymer.
[0104] In one embodiment, the second agent may be capable of
enhancing tubulin acetylation. In another embodiment, the second
agent may be capable of enhancing the sensitivity of the cell to a
therapeutic agent and/or may be capable of modifying the host
genetic status.
[0105] In one embodiment, the aforementioned second agent may be
selected from a histone deacetylase inhibitor (HDACi), a tubulin
binding agent (TBA) and siRNA that is capable of directly or
indirectly affecting the microtubule network stability. The HDACi
may be selected from Tubastatin A, belinostat, bufexamac,
panobinostat, PCI-24781, SAHA (vorinostat), scriptaid, trichostatin
A, valporic acid, B2, salermide, sirtinol and combinations
thereof.
[0106] In one example, it is shown that the TBA of the second agent
of the present invention may be selected from taxanes, epothilones,
and a combination thereof. The taxanes may be paclitaxel, docetaxel
or a combination thereof.
[0107] In a further example, the taxanes may be selected from a
group consisting of cremophor EL.RTM. Taxoprexin.RTM.
(Docosahexaenoic acid-paclitaxel), Xytotax.TM. (paclitaxel
polyglumex), TOCOSOL.RTM. paclitaxel, BMS-184476, DJ-927,
BMS-275183, RPR 109881A, Ortataxel, Genexol (co-polymer
combination), LEP (liposomal-encapsulated paclitaxel) and taxol in
vitamin E emulsion.
[0108] In one example, the epothilones may be patupilone,
ixabepilone, BMS 310705, sagoilone, KOS-862, KOS-1584, or
combinations thereof.
[0109] In one embodiment, the chemosensitizers may be histone
deacetylase inhibitors (HDACi), tubulin binding agents, taxanes and
siRNA, capable of directly or indirectly affecting the microtubule
network stability.
[0110] Advantageously, the chemosensitizers used in the present
disclosure are known to have anti-neoplastic, anti-inflammatory,
anti-angiogenic or neuroprotective effects in vivo.
[0111] In one embodiment, the genetic material may be coupled to at
least one cationic species. The cationic species may be selected
from polyethylene imine, polycationic amphiphiles, DEAE-dextran,
cationic polymers, their derivatives and combinations thereof.
Furthermore, the cationic species may be a cationic polymer such as
a dendimer, branched-polyethylenimine (BPEI),
linear-polyethylenimine (LPEI), Poly(amindoamine) (PAMAM),
XtremeGENE HP.RTM., and combinations thereof. In one embodiment,
wherein the cationic species is LPEI.
[0112] In one embodiment, the nucleic acid:polymer (N/P) ratio
cationic species to the genetic material may be selected from the
group consisting of from about 0 to about 1000, about 0 to about
900 about 0 to about 800 about 0 to about 700 about 0 to about 600
about 0 to about 500 about 0 to about 400 about 0 to about 300
about 0 to about 200 about 0 to about 100 about 0 to about 75 about
0 to about 50 about 0 to about 25 between about 0 to about 15,
between about 0 to about 10, and between about 0 to about 5. In one
embodiment, the nucleic acid:polymer (N/P) ratio of genetic
material to cationic species may be 20.
[0113] In one embodiment, the N/P ratio is the ratio of the
cationic species to the genetic material forming polyplexes within
the composition.
[0114] In another embodiment, the genetic material may be a nucleic
acid sequence. In another embodiment, nucleic acid sequence may be
selected from the group consisting of DNA, RNA, mRNA, ribozymes,
antisense oligonucleotides, modified polynucleotides and
combinations thereof.
[0115] In one embodiment, the cell is a differentiated or
undifferentiated cell. In one embodiment, the cell may be selected
from the group consisting of nervous systems cell, liver cell,
hematopoiesis cell, peripheral blood cell, umbilical blood cell,
bone marrow cell, tumour cell, ischemic tissue cell, T cells, B
cells, skin cells and combinations thereof.
[0116] In one embodiment, the cell may be isolated from a
biological sample. In one embodiment, the biological material may
be selected from the group consisting of a sample of fresh tissue,
frozen tissue, paraffin-preserved tissue and/or ethanol preserved
tissue. In another embodiment, the biological material may be
selected from the group consisting of whole blood or a component
thereof, lymph, bile fluid, cerebrospinal fluid, bronchioalveolar
lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast
milk, amniotic fluid, a buccal smear and pus.
[0117] In one embodiment, the first agent and the second agent may
be provided to the cell simultaneously, separately or sequentially.
In one embodiment, the first agent and the second agent may be
provided within the first hour of transfection of the cell with the
genetic material. Alternatively, the first agent and the second
agent may be provided within about 2, 3, 4, 5, 6, 7, 8, 9, 10 hours
of transfection of the cell with the genetic material
[0118] In one embodiment, the second agent may be provided after
the first agent. In one embodiment, the second agent may comprise a
two or more therapeutic agents as described herein.
[0119] In one embodiment, the composition as described herein may
be used in gene therapy.
[0120] In one embodiment, the composition as described herein may
further comprise a therapeutic agent. The therapeutic agent may be
chemotherapeutic agent. In one embodiment, the chemotherapeutic
agent may be selected from the group consisting of taxanes,
paclitaxel, docetaxel and epothilones, and combinations
thereof.
[0121] In one embodiment there is provided a method of treating a
patient in need of gene therapy comprising administering a genetic
material and a composition as described herein.
[0122] In another embodiment, there is provided the use of a
composition as described herein in the manufacture of a medicament
for treating a disease, selected from a group consisting of cancer,
SMA, bone cancers, leukemia, blood cancers, sickle cell disease,
Wiskott-Aldrich Syndrome, HIV, genetic disease, diabetes,
monogenic, infectious neurological, ocular, inflammatory, cardiac
and neurodegenerative diseases.
[0123] In a further embodiment, the composition as described herein
may be used for gene marking.
[0124] In one embodiment, there is provided a method of delivering
a genetic material into a cell comprising the step of administering
the genetic material with the composition as described herein. The
method may be performed in vitro or ex vivo or in situ.
[0125] In one embodiment, here is provided a composition comprising
a first agent, as defined herein, and a second agent, as defined
herein.
[0126] In another embodiment, there is provided a kit comprising a
first agent, as defined herein, and a second agent, as defined
herein, a therapeutic agent as defined herein and instructions for
use.
[0127] The present disclosure further encompasses therapeutic genes
or genetic material. As exemplified in the examples below, this
genetic material may comprise of, but is not limited to, nucleic
acid sequences, encoding for any one gene or part thereof, which
upon entering the cell having then a therapeutic effect upon the
target gene by possibly enhancing expression or by possibly
decreasing expression by interacting with the target sequence. It
should be understood that the genetic material, including
therapeutic genes, may differ from any exact sequences illustrated
and described herein. Thus, the invention contemplates deletions,
additions and substitutions to the sequences shown, so long as the
sequences function in accordance with the methods of the invention.
For the purpose of the present invention, any method of quantifying
genetic material is appropriate for use in the context of the
invention and many are known in the art. For example, the genetic
material may be isolated using techniques known to the art, e.g.
restriction enzymatic digest, polymerase chain reactions (PCR),
agarose gel electrophoresis, size exclusion chromatography and many
more. Furthermore, the isolated genetic material may be quantified
using methods known in the art, that may be, but are not limited
to, spectrophotometric analysis, fluorescent labelling,
quantitative PCR (qPCR, also known as real-time PCR), and e.g. DNA
microarray (CHIP) analysis for the determination of gene expression
levels after transfection. All the methods disclosed may be used
consecutively in any possible order.
[0128] The composition as described herein may be used in many
different applications, all of which have/pertain to the use of
TrafEn.TM. as a unique approach, wherein the simultaneous effects
of both genes and drugs (e.g., HDACi) with microtubule modifying
activities and/or epigenetic modifying activities may be useful for
all the areas described below.
[0129] Cell therapy refers to the process of introducing cells to
restore normal function; which was lost due to age, disease,
damage, or congenital defects. There are many forms of cell-based
therapy, including stem cells and immune cells and cancer cells.
Additionally, HDACi is known to effect processes in stem cell
differentiation and reprogramming of somatic cells into induced
pluripotent stem cells (iPSCs), broadening the application of
TrafEn.TM. in cell based therapy. These cell based therapies may be
utilised, but are not limited to, the application of stem cells,
e.g. negative or positive selection for the segregation of modified
populations, and/or controlling the growth of stem cells and their
progeny, e.g. when inserting a suicide gene into the stem cells
population to enable the use of external stimuli to eradicate
uncontrolled cell proliferation. Further applications for this
invention may also be modifying stem cells to delivery gene
product, which depending on the application, may require long-term
or transient expression of therapeutic gene. Cases like wound
healing, bone regeneration, angiogenesis, and repair of central
nervous system injury, transient expression would be preferable
examples. On the other hand, to correct genetic disease, persistent
gene expression is required.
[0130] Another possible application of this invention is modulating
differentiation in stem cells via a transfer of genes mediated by
this invention. Another potential application of gene transfer to
stem cells is to provide genetic signals, improving the outcome of
differentiation protocols know in the art. The possibilities of
directing cell differentiation through gain--(plasmid DNA) or
lost--of function (shRNA, siRNA, miRNA), lead to a more pure
population of differentiated cells, thus becoming a particularly
attractive example of this invention, as the clinical applicability
of differentiating and reprogramming cells in cell based therapy is
known to be driving the exploration of the use of various types of
stem cells, which may be, but are not limited to, induced
pluripotent stem cells, mesenchymal stem cells, neural stem cells,
iPSCs, ESCs, human embryonic stem cells (hESCs), ASCs,
hematopoietic (HSCs) and mesenchymal stem cells (MSCs), Neural stem
cells (NSCs), immune cells (boosting of T-cell function), cancer
cells (for cancer vaccinations) and genetically modified variations
thereof, neurons, microglia, astrocytes
[0131] Possible applications of the composition as described herein
for the treatment and therapy, e.g. cell based and gene
therapy/therapeutics of any one of, but not limited to, the
following diseases: SMA (via iPS cell based therapy), bone cancers,
leukemia, blood cancers, cancers (anti-angiogenic, stopping cell
growth), HSC therapies for sickle cell disease and Wiskott-Aldrich
Syndrome, gene treatment for cancer, HIV infection and genetic
disease, repair damaged tissues, diabetes (T1D and T2D), cardiac
disease, neurodegenerative diseases, for example, Parkinson's
disease.
[0132] The composition as described herein may also be used
additionally as a method for reprogramming cells, and or
transdifferentiation of cells. Genetic tools known in the art have
been established to initiate reprogramming process in various cell
types.
[0133] Furthermore, it has been suggested that HDACi may play a
possible role in promoting transdifferentiation. It has been shown
in the art that HDACi induced expression of SOX9 in hepatocytes,
which normally lack SOX9. The aberrant expression of SOX9 induced
expression of COL2A1 and COMP1, which is usually found during
chondrogenesis. These observations demonstrate redeployment of a
typical developmental process to an atypical setting may be
initiated by treatment of cells with HDACi.
EXPERIMENTAL SECTION
Example 1
Mild Centrifugation Improved Transfection Efficiency and Reduced
Cytotoxicity in Neuronal Cells
[0134] To establish an optimal protocol for transfection of
neuronal cells, cellular toxicity to bolus transfection, where the
cells were exposed to a defined composition of polyplex for a
period of time, was first investigated. Increasing N/P ratios of
polyplex was found to reduce cell viability of native Neuro2A and
NG-108 cells even in the presence of serum (FIG. 11). We
hypothesized that cytotoxicity at high N/P ratio was due to
prolonged exposure of cells to toxic free polymer, and shortening
the period of incubation should reduce toxicity. However,
transfection efficiency was significantly reduced with shorter
periods of incubation (15 min or 1 h, FIG. 12a). Mild
centrifugation was previously reported to improve transfection.
Next, this approach was explored as an attempt to improve
transfection at short incubation periods. As hypothesized, shorter
incubation (15 min) coupled with mild centrifugation resulted in
transfection efficiency comparable to bolus transfection (FIG.
12b). As previously reported, it was found that LPEI/pDNA polyplex
aggregate and deposit extensively in salt containing physiological
media (FIG. 13), which may explain the beneficial effect of mild
centrifugation in transfection.
Example 2
Polyplex Formed Aggregates and were Sedimented by Mild
Centrifugation
[0135] Expectedly, dynamic light scattering studies showed that
nanosized LPEI/pDNA (.about.100 nm) rapidly increased in size when
placed in DMEM (FIG. 13a), consistent with low colloidal stability
and the formation of aggregates in high salt conditions (Wightman,
L., et al. J Gene Med, 2001. 3(4): p. 362-72; Mishra, S., P.
Webster, and M. E. Davis. Eur J Cell Biol, 2004. 83(3): p. 97-111).
The deposition of these large aggregates over time was likely to
account for the presence of distinct particulates found on the
surface of cell culture plates after the incubation of LPEI/pDNA
polyplexes in Dulbecco's minimum essential medium DMEM (but not in
HEPES) (FIG. 13b). These large heterogeneous particulates (>0.5
.mu.M) on the surface of the wells were observed even in the
absence of cells, ruling out the possibility of cellular artefacts.
In order to quantify the amount of pDNA in the supernatant, pDNA
was efficiently released from the polyplexes and measured by qPCR.
After centrifugation, the amount of pDNA in the supernatant was
significantly reduced, indicative of the sedimentation of
polyplexes in DMEM (FIG. 38). Collectively, these observations were
indicative of the sedimentation of aggregated polyplexes onto the
substrate over time and the deposition of these aggregates was
enhanced by mild centrifugation.
Example 3
Centrifugation Enhanced Transfection Resulted in Efficient Gene
Delivery into Native but not Differentiated Neuronal Cells
[0136] This approach was then applied to transfect differentiated
Neuro2A and NG-108 cells. Both cell-lines were pharmacologically
induced to differentiate and showed elaborate neurite outgrowths
prior to transfection (FIG. 5b). Intriguingly, both differentiated
cell types were poorly transfected as compared to the same native
cells (FIG. 5a, b). Indeed, most of the EGFP positive cells
demonstrated undifferentiated phenotype, where only .about.3% of
the EGFP positive cells were found to bear neurites twice the body
length (FIG. 5c). Increasing the N/P ratio (from 10 to 50) did not
increase the efficiency of transfection. Similar to native cells,
negligible toxicity was observed in differentiated cells
post-transfection (FIG. 14). Collectively, these observations
demonstrated that differentiated neuronal cells were refractory to
transfection with LPEI/pDNA polyplex as compared to the matched
native cells.
Example 4
Cellular Binding and Internalization of Polyplex was not Affected
after Neuronal Differentiation
[0137] To assess if DNA uptake was affected upon neuronal
differentiation, the total amount of DNA associated with the cells
was measured by quantitative real-time PCR (qPCR) in differentiated
and native cells. To quantify the amount of pDNA associated with
cells, pDNA was efficiently released from the polyplex and measured
by quantitative real-time PCR (qPCR) (FIG. 15b, c). The lysis
solution used to release pDNA from polyplex did not affect the qPCR
reaction (FIG. 15a). Comparable amounts of DNA (.about.10.sup.6
copies/cell) were associated with native and differentiated cells
over time (FIG. 16), indicative that the polyplex associated
equally well regardless of the differentiation state of the cells.
To examine whether the polyplex were internalized in differentiated
cells, cells were transfected with Rhodamine-pDNA. The fluorescence
of extracellular polyplex was effectively quenched using trypan
blue (FIG. 17). Intriguingly, intracellular fluorescent signal
intensities were detected at significant levels in both native and
differentiated cells (FIG. 6a). This observation was further
confirmed by qPCR, showing comparable amounts of pDNA internalized
in native and differentiated cells 4, 24 and 48 h post-transfection
(FIG. 6b). Extracellular pDNA was effectively removed by pAA/DNase
treatment prior to qPCR measurements (FIG. 18). These results
suggested that cellular association/uptake of aggregated polyplex
were not significantly different after neuronal
differentiation.
Example 5
Polyplex Internalization in Native, but not Differentiated Neuronal
Cells, Involved PKC
[0138] To test the hypothesis that distinct biochemical mechanisms
may be involved in the uptake of polyplex in native and
differentiated cells, we examined the contributions of various
signalling pathways pharmacologically. Dynasore, an inhibitor of
dynamin GTPase activity, significantly inhibited the uptake of
polyplex in differentiated and native Neuro2A cells. Filipin III, a
cholesterol sequestration agent, did not affect polyplex uptake in
either phenotypes. Intriguingly, Rottlerin, a protein kinase C
inhibitor, reduced the uptake of polyplex significantly in native
but not in differentiated cells (FIG. 7a). The effects of these
inhibitors on expression of the transgene were similarly correlated
(FIGS. 7b and c). The effect of PKC inhibition on transfection was
further confirmed with another PKC inhibitor, GO6983 (FIG. 19).
Similar observations were also made with NG-108 cells (FIG. 20).
The differential involvement of PKC in the internalization of
polyplex is consistent with the suggestion that cellular
trafficking mechanisms have altered upon neuronal
differentiation.
Example 6
Sedimented Polyplex were Bioavailable and Mediated Efficient
Transfection
[0139] Particle size is thought to affect endosomal uptake,
intracellular transport and nuclear entry, and the size dependency
may differ in different cell types and applications. To address the
contribution of size to transfection, pre-complexed LPEI/pDNA in
DMEM was first passed through a 0.22 .mu.m filter. The filtrates
were then used to transfect cells (see experimental design in FIG.
39). Interestingly, the removal of polyplex aggregates from the
transfection solutions almost completely abolished transfection
(FIG. 40a). In contrast, filtrates of LPEI/pDNA pre-complexed in
HEPES (average size of polyplex<200 nm) could efficiently
transfect cells after incubation in DMEM (FIG. 41). Furthermore, a
second filtering step upon incubation of HEPES filtrates in DMEM
abolished the transfection. These findings supported the hypothesis
that polyplexes aggregate only in high salt medium and are critical
for efficient transfection. In parallel, the amount of pDNA in the
filtrates were significantly reduced, suggesting that large,
aggregated polyplexes (>200 nm) has been effectively removed by
filtration (FIG. 42). Next, the hypothesis was tested that
suggested aggregated polyplexes deposited on the surface of the
substrate could efficiently transfect cells. Aggregated polyplex
was first sedimented by mild centrifugation onto the surface of the
wells, the supernatant removed and the wells were washed with
media. Neuro2A cells were then seeded directly onto these surfaces
pre-loaded with sedimented polyplex aggregates. Interestingly, the
cells were efficiently transfected, suggesting that these
sedimented aggregates were highly bioavailable (FIG. 40a) and the
percentage of cells transfected correlated to the amount of pDNA in
the polyplex (FIG. 40b). The above observations led us to the
question as to how such large aggregates were internalized by
cells. It has been proposed that surface immobilized LPEI/pDNA
polyplexes mediate transfection by releasing significant amounts of
nanosized polyplexes into the media, thus allowing the polyplexes
to be internalized by the cells. To test the hypothesis that
surface deposited aggregates release significant amounts of pDNA
into the media, the distribution of pDNA in the culture system was
measured over a period of 48 h. In the presence or absence of
cells, the amount of pDNA released into the media was <1% of the
total amount of pDNA found in the well throughout the 48 h period
of incubation, suggesting that pDNA was not efficiently released
from the surface bound aggregate (FIG. 43).
Example 7
Intracellular Plasmid DNA was Localized to Acidic Compartment in
Differentiated Neuronal Cells
[0140] A comparison of the intracellular behaviours of LPEI/DNA
polyplex and adenovirus in primary neurons demonstrated that the
polyplex, but not adenovirus, was found to be sequestered in acidic
compartment, possibly accounting for the poor transfection
efficiency of polyplex. As transfection was found to be highly
efficient in native but not differentiated cells (FIG. 5a), we
hypothesized that changes in intracellular trafficking of polyplex
may be a critical reason. To test this hypothesis, we exploited the
pH sensitive property of fluorescein to examine the intracellular
localization of polyplex.
[0141] After extracellular fluorescence was quenched by ethidium
bromide (EtBr), distinct fluorescence was observed in majority of
native cells but significantly reduced in differentiated cells when
transfected with polyplex containing FITC-pDNA (FIG. 8a). To verify
that pDNA was taken up into the cells, polyplex containing
Rhodamine-pDNA (pH insensitive) was similarly transfected into both
native and differentiated cells. Rhodamine-pDNA was observed in
majority of both native and differentiated cells; further
supporting the observation that both phenotypes internalized
polyplex efficiently (FIG. 6). Percentage of cells containing FITC-
or Rhodamine-pDNA was comparable in native cells while FITC/Rho
ratio was significantly lower in differentiated cells; indicating
quenching of intracellular FITC (FIG. 8b). Taken together, these
data suggested the sequestration of polyplex in acidic compartments
in differentiated but not native cells. To test whether this is a
general phenomenon, similar experiments were performed on
differentiated NG-108 cells as well as primary cortical neurons.
Consistently, quenching of FITC was observed (FIGS. 21 and 22);
suggesting polyplex trafficking to acidic compartment may be an
important factor contributing to the poor transfection of
differentiated neurons.
[0142] To validate the trafficking of pDNA to acidic compartment in
differentiated cells, co-localization of pDNA with lysosensor green
labelled acidic compartment was visualized using confocal imaging.
Localization of Rhodamine-pDNA to the acidic compartment was
observed 4 h post transfection and increased over time (24 h) in
both Neuro2A (FIG. 8c, d) and NG-108 cells (FIG. 23). It is worthy
to note that the total pixel count of labelled acidic compartment
per cell was significantly higher in differentiated cells (FIG.
24). These results suggested that intracellular trafficking of
polyplex to acidic compartments directly or indirectly in
differentiated neurons may in part contribute to the poor
transfection efficiency.
Example 8
Escape of Polyplex from Acidic Compartment Enhanced Transfection in
Differentiated Cells
[0143] It was hypothesized that facilitating the escape of polyplex
from the acidic compartment may increase transfection efficiency.
Addition of pH sensitive DOPE/CHEMS resulted in a 10-fold
augmentation of transfection efficiency in differentiated neurons
(FIGS. 9a and b). To investigate the fusogenic effect of
DOPE/CHMES, trafficking of polyplex to acidic compartment was
examined with labelled pDNA. As expected, the amount of DNA
localized in the acidic compartment was drastically reduced after
treatment with DOPE/CHEMS (FIGS. 9c and d), similar to the
intracellular localization profile observed in native cells.
Real-time tracking of the release of polyplex from the compartment
(FIG. 25) clearly revealed the ability of DOPE/CHEM in facilitating
the escape of polyplex from the acidic compartments. In addition,
the effect of DOPE/CHEMS was found to be temporal, where the
optimal effect was achieved when the chemical was added immediately
after centrifugation (FIG. 26). Evidently, the localization of
polyplex to acidic compartment in differentiated neurons led to
poor transfection, which can be improved by enhancing endosomal
escape of polyplex using fusogenic lipids.
[0144] It is also worthy to note that chloroquine, a lysosomotropic
compound known to inhibit fusion of the endosome and reduce
enzymatic degradation of DNA by buffering the vesicular interior,
did not show cumulative enhancement of transfection. Similar
observations were found with PLUS.TM. reagent and INF7 fusogenic
peptide (FIG. 27) when these reagents were added to the culture
post-transfection. Additionally, LPEI mediated delivery of pDNA
pre-complexed with PLUS reagents and INF7 fusogenic peptide did not
improve transfection in differentiated neuronal cells (FIG.
28).
Example 9
Enhanced Microtubule Mediated Trafficking and Endosomal Escape of
Polyplex Enabled the Efficient Transfection of Differentiated
Cells
[0145] Microtubule mediated transportation is known to play a role
in the polymer/pDNA trafficking to the nucleus. Armed with the
capability in releasing polyplex from the acidic compartments, we
next explored the influence of chemotherapeutic mediators of
intracellular trafficking as a strategy to further enhance
transfection efficiency. In particular, Tubastatin A, a histone
deacetylase-6 (HDAC6) inhibitor that enhances microtubule mediated
intracellular transport of cargo, was evaluated. The
co-administration of DOPE/CHEMS and Tubastatin A resulted in a
highly significant increase in the number of EGFP positive
differentiated Neuro2A (.about.70%) (FIGS. 10a and b). A time
course study revealed that minimal exposure of cells to DOPE/CHEMS
and Tubastatin A for 12 h is required for high transfection
efficiency (FIG. 29). Interestingly, the combinatorial effect of
DOPE/CHEMS and Tubastatin A was found to be carrier dependent,
enhancement in transfection occurred when LPEI, PAMAM, and
XtremeGENE XP but not Fugene HD was used (FIG. 30). Additionally,
Trichostatin A, an alternative HDAC inhibitor (HDACi), greatly
enhanced transfection efficiency in the presence of DOPE/CHEMS
(FIG. 31). Next, the strategy of mild centrifugation to sediment
aggregated polyplex with the co-administration of DOPE/CHEM and
Tubastatin A, was evaluated on primary cortical neurons.
Remarkably, close to 75% of these post-mitotic cortical neurons
were transfected (FIGS. 10c and d) without significant cytotoxicity
(FIG. 32a). It should be noted that optimal transfection occurred
only when reagents were co-administered within the first hour of
transfection (FIG. 32b). To further confirm the significance of
microtubular stabilization for efficient transfection, the effect
of paclitaxel and various HDACi on tubulin acetylation and
transfection was examined. In line with previous reports, HDAC6
targeting small molecules (Tubastatin A, TSA, Varinostat/SAHA) and
paclitaxel but not Entinostat, Tacedenaline markedly enhanced
tubulin acetylation (FIG. 33). Additive effect on transfection was
observed when cells were treated with DOPE/CHEMS and small
molecules that induced tubulin acetylation (FIG. 34). All together,
these data suggested the endosomal escape and microtubular
stabilization enhanced transfection through a concerted
mechanism.
[0146] Enhancement of transgene expression is achieved by
TrafEn.TM. combinations acting on the endosomal trafficking and the
stabilization of microtubular network. The effect of TrafEn.TM. on
transfection enhancement has been examined in various conditions
including--cell types, gene carriers, genetic material and
HDACi.
Example 10
Effect of TrafEn.TM. Differed in Various Cell Types
[0147] It is known that various cell types and even cell lines from
the same tissue origin displayed differential transfection
efficiencies. Extending the TrafEn.TM. principle findings,
significant enhancements of transfection were also observed with
several cell types (FIG. 44), lending further evidence that the
coordinated endosomal release and the stabilization of microtubule
network contribute significantly to transfection efficiency.
Additionally, this approach can produce cell line models of the
same tissue of origin with comparable expression levels of
transgene by controlling transfection efficiencies.
Example 11
TrafEn.TM. Enhanced Transfection of Various Commercial Gene
Carriers
[0148] The combinatorial effect of DOPE/CHEMS and Tubastatin A was
also found to enhance the transfection efficiencies of a number of
commercially available polymers beside LPEI (FIG. 45). All
commercial reagents, except XtremeGene HP, performed poorly when
the transfection experiment was conducted with manufacturer's
protocol. Transfection enhancements were observed when the
manufacturer's protocol was replaced with the deposit mediated
transfection workflow. Further enhancement was achieved with
addition of TrafEn.TM. reagent. Enhancement of transfection by
facilitating endosomal escape and microtubule trafficking of the
various polymers suggested the transfection mechanisms of different
carriers may be similar.
Example 12
TrafEn.TM. Enhanced Transfection of shRNA
[0149] Genetic manipulations may be accomplished by either gene
overexpression using plasmid DNA (pDNA) or gene knockdown with
short hairpin RNA (shRNA). To date, a library of shRNA had been
designed to suppress the expression of desired genes in mammalian
cells. For example, repression of PTB with shRNA was reported to
elicit cellular reprogramming and transdifferentiate a multiple
cell types to neuronal like cells. To demonstrate the TrafEn.TM.
effect on gene knockdown, a case study showing efficient repression
of the RNA binding polypyrimidine-tract-binding (PTB) protein in
the presence of TrafEn.TM. is presented. The mRNA level of PTB1 in
HeLa cells (cervical cancer cell line) were examined after
transfection. Evidently, the knockdown efficiency is more efficient
in the presence of TrafEn.TM. (Bars 1 to 4, FIG. 46). The efficient
gene knockdown contributed to the rapid transdifferentiation
process where neuronal like cells were observed after 8 days of
treatment (FIG. 47).
Example 13
Gene-Drug Combinatorial Strategy
[0150] By coupling the effect of the time-dependent rerouting from
non-productive endosomal pathway using DOPE/CHEMS with a
microtubular network stabilizer HDAC6 inhibitor, enhancement of
transfection has been demonstrated in a number of cell types. Some
HDAC inhibitors such as Trichostatin A (TSA) and Varinostat (SAHA)
enhance transfection by targeting microtubule and at the same time
exert epigenetic modification activities. The effects of these
substances on the histone and non-histone acetylation status have
implications in various clinical applications. For example, HDACi
has been used as chemosensitizer to increase cytotoxic effect of a
therapeutic agent for cancer (A), to induce differentiation of stem
cell (B), to increase immunogenicity of cancer vaccine (C) and to
enhance reprogramming process (D). The realization of the broad
clinical applications of HDACi has led to studies which explore the
possibility of a drug-gene combinatorial effect DOPE/CHEMS and
HDAC6 inhibitor work together to enhance transfection efficiency (1
and 2, FIG. 1) and HDACi may augment the effect of the transgene
through epigenetic modification (3, FIG. 1). In other words, the
gene-drug combinatorial strategy simultaneously increases transgene
expression and the synergistic effect/s of HDACi may further
accentuate the therapeutic effect.
Example 14
Synergistic Effect of p53 Overexpression and SAHA to Induce High
Cytotoxicity
[0151] It is known that treatment with single anti-cancer agents
such as gene therapy and HDACi alone often demonstrate limited
clinical benefit for patients with solid tumour. For instance,
anti-cancer regime with single therapeutic agent such as p53
replacement therapy and SAHA has shown poor clinical outcome,
prompting the development of novel combination regime with other
cancer therapeutics. p53 is known to be involved in numerous
functions including the regulation of cell cycle, DNA repair and
activation of apoptosis. The efficacy of p53 replacement therapy
has been tested in glioma, which 30-60% of this cancer were found
to display mutations in p53. Despite the many preclinical and
clinical studies conducted to date, none has progressed beyond
phase I trials. On the other hand, SAHA has shown moderate clinical
benefits as monotherapies but has garnered much excitement in
combination therapy. SAHA has been shown to exert potent
anti-tumour effects in a broad variety of cancer cells at
concentrations that have minimal toxic effects on normal cells.
Although the underlying anticancer mechanisms of SAHA are still
unclear, it is likely that it exerts epigenetic modification that
altered gene expressions resulting in growth arrest, migration
inhibition and cell death. A study has shown SAHA induced
up-regulation of pro-apoptotic genes (Bax, Bim, Bmf, Bik,
cytochrome C and Smac) and down-regulation of anti-apoptotic genes
(XIAP and survivin). Other study has shown that the susceptibility
of SAHA induced cell death was regulated by p53. These observations
suggest a possible synergistic effect of SAHA with p53, and thus,
suggest a possible combinatorial gene-drug effect. To illustrate
the synergistic effect of the gene-drug combination (TrafEn.TM.),
the therapeutic outcome of combined p53 and HDACi treatment was
examined in U251MG glioma cell line (U251MG). The strategy
presented here demonstrates the combinatorial effect of enhanced
p53 transfection efficiency by DOPE/CHEMS and SAHA, which the
latter compound may also produce an epigenetic effect. First, the
effect of DOPE/CHEMS and SAHA on transfection enhancement was
confirmed in U251MG glioma cells (p53 mutant) (FIG. 48). Following
transfection, treatment of cells with single or combination of the
various reagents (DOPE/CHEMS, Tubastatin A and SAHA) resulted in
significant enhancement in transfection efficiency. Interestingly,
in all cases, the percentage (%) of cells expressing high level of
GFP (as determined by the RFU) was increased significantly in the
presence of transfection enhancers. The co-administration of
DOPE/CHEMS and Tubastatin A/SAHA displayed superior effect over
treatment with single reagent. Next, the combinatorial effect of
p53 with the transfection enhancers was examined. Compared to the
negative control (-ve), the cell viabilities were affected to
various extent (FIG. 49). Significant reduction in cell number was
observed with p53 but not PMAXGFP overexpression, suggesting that
cell death was p53 dependent (possibly through apoptotic pathway as
suggested by other studies. Further increment of cell death with
p53 was found in the presence of DOPE/CHEMS, a non-toxic fusogenic
lipid, thus, providing evidence of enhanced killing of cells by
improving transfection efficiency. While transfection efficiencies
of U251MG treated with DOPE/CHEMS or SAHA were comparable (FIG.
48), combination of p53 and SAHA resulted in more cell death of
U251MG cells. This observation is consistent with the reported
chemosensitizing effect of SAHA on anti-cancer action.
Intriguingly, p53 delivery to cells in the presence of DOPE/CHEMS
and SAHA exhibited superior cytotoxic effect (with .about.95%
reduction of cell number) over other conditions where transfection
enhancers were added individually. This data supported our
hypothesis that the anti-cancer drug and transgene product
synergise with each other, augmenting the therapeutic outcome.
Furthermore, increasing the duration of incubation with SAHA did
not further reduce the cell number (FIG. 50). Next, the effect of
Tubastatin A (cytosolic HDAC6 specific inhibitor) was compared with
SAHA to further confirm the contribution of the epigenetic
modulation of SAHA on p53 induced cell death (FIG. 51). Unlike
SAHA, Tubastatin A did not result in greater reduction of cell
number as compared to DOPE/CHEMS. As expected, transfection of p53
with Tubastatin A and DOPE/CHEMS did not contribute to the
drug-gene combinatorial effect as observed with the use of
SAHA.
[0152] Accumulating evidence suggests that glioma heterogeneity
likely is the key reason to treatment failure. To date, two
subpopulations, known as TMZ resistant cells and cancer stem cells,
have been identified. Both of the subpopulations have developed
genetic mechanisms that exhibited aggressive cancer phenotypes such
as migration and resistance to TMZ. The cancer stem cells
hypothesis is well recognized as a challenge for treatment, due to
resistance. These cells exhibit stem cell-like characteristics and
are capable to generate heterogeneous tumour masses. Recent studies
support the presence of small number of cancer stem cells in glioma
(GSC). As these cells are resistant to radiotherapy and
chemotherapy, they are sufficient to generate recurrent tumour.
Next, the synergistic effect of the gene-drug combination using
p53, DOPE/CHEMS and SAHA was examined in U251MG cells resistant to
40 .mu.M TMZ (TMZR-U251MG) and GSC (FIG. 52). Evidently, the
strategy has superior cytotoxic effect, resulting in 89.1% and
91.7% reduction in cell number of TMZR-U251MG and GCS respectively.
Interestingly, overexpression of p53 alone resulted in significant
cell death in TMZR-U251MG but has no effect in GSC. This has not
been reported and is the first time that the effect of
overexpression of p53 was examined in the parental, TMZ resistant
cells and GS.
Example 15
Synergistic Effect of BMP2 Overexpression and TSA to Induce
Osteogenic Differentiation
[0153] To realize the clinical application of stem cells,
technologies must be established, to direct stem cells to
differentiate in a regulated manner, to circumvent immunogenicity
of non-autologous stem cells-derived cells and to function as drug
delivery vehicles and to regulate the growth of cells
post-transplantation. Gene delivery is a potential tool to deliver
biological signals to address these challenges. The applications of
TrafEn.TM. gene-drug combination in stem cells technologies was
illustrated with mesenchymal stem cells (MSCs).
[0154] To examine the drug-gene combination effect, we first
examined the effect of TSA (HDAC6 inhibitor and epigenetic
modifier) in transfecting MSC (FIG. 53). The addition of DOPE/CHEMS
and TSA individually led to significant enhancement of transfection
but no further enhancement was observed in when used in
combination. Next, the expression levels of BMP2 in the presence or
absence of DOPE/CHEMS and/or HDACi (TSA, Tubastatin A) in MSC were
quantified using qPCR to test a drug-gene combinatorial effect
(FIG. 54). Significant increase in the expression level of BMP2 was
observed 3 day post transfection (.about.13,000 fold over the
negative control) with PMAXGFP-BMP2 (BMP2). The addition of
DOPE/CHEMS (BMP2+DOPE/CHEMS) or Tubastatin A (BMP2+5 .mu.M
Tubastatin A) enhanced BMP2 expression by .about.20,000 fold.
Surprisingly, treatment with TSA did not further enhance BMP2
expression (BMP2+150 nM TSA). Intriguingly, combination of
DOPE/CHEMS and TSA elicited a far superior effect on BMP2
overexpression (.about.200,000 fold as compared to control) than
the combination of DOPE/CHEMS and Tubastatin A (.about.80,000
fold). These unanticipated results are suggestive of a distinct
function of TSA beyond merely stabilizing the tubulin network in
enhancing BMP2 expression and that this can serve as an opportunity
to identify novel effects of gene-drug combinations by using
specific HDACi. It is worthy to note that prolonged expression of
transgene of up to 21 day in MSC culture was observed with cells
were treated with DOPE/CHEMS and TSA (FIG. 55). This finding
indicates potential efficient cell therapy with prolonged released
of therapeutic product. While further characterization and
functional analysis are required to validate BMP2 release and
osteogenesis, we observed extensive calcium deposition at the end
of the study in cells maintained in osteogenesis defined media
(FIG. 56B) but not in expansion media (FIG. 56A).
Example 16
Synergistic Effect of GM-CSF Overexpression and SAHA to Improve
Immunogenicity of Cancer Cells
[0155] Cancer vaccine can be generated from the genetically
modified tumour cells, either autologous (removed from patients
during surgery) or allogeneic (established cancer cell lines).
After genetic modification, cells are inactivated by radiation and
injected to the patients subcutaneously or intradermally to induce
recipient's immune response against the tumour cells. Utilization
of allogeneic cells, such as existing cell lines, provides a
sustained and unlimited source of well-characterized cells, which
can be standardized for large-scale production. It may also provide
single batch for clinical lot for comparative analysis of clinical
result and eliminates the need to continuous harvest patient's
cancer cell. Furthermore, there is no longer the requirement of
tailor-made individual cancer vaccine, which may increase cost and
labour. To date, the most advanced cell line based cancer vaccine
is GVAX, comprising PC3 cells modified with GM-CSF gene for
metastatic prostate cancer treatment. Modified PC3 cells are able
to activate antigen-presenting cells (APC) and induce immune
response. HDACi, on the other hand, can augment the immunogenicity
of tumour cells by increasing expression of major
histocompatibility complex (MHC) class I and II proteins,
co-stimulatory/adhesion molecules. Moreover, treatment with HDACi
such as SAHA and TSA has been found to associate with enhanced
presentation of MICA and MICB on the surface of cancer cells but
not normal cells. MICA and MICB induce activation of
immunoreceptor, natural killer cell protein group 2D (NKG2D),
resulting in the increased susceptibility of cancer cells to
natural killer cells (NK cells; CD4 and CD8 T cells). Here, we aim
to examine the combination of GM-CSF and SAHA in augmentation of
the immunogenicity of PC3 cells. This TrafEn.TM. gene-drug strategy
may generate PC3 cancer vaccines, that are capable to activate both
APC and NK cells.
[0156] The effect of SAHA (HDAC6 inhibitor and epigenetic modifier)
in transfecting PC3 was first examined (FIG. 57). The addition of
DOPE/CHEMS, Tubastatin A and TSA individually led to significant
enhancement of transfection but no further enhancement was observed
in when used in combination. Next, the expression levels of GM-CSF
in the presence or absence of DOPE/CHEMS and/or SAHA in PC3 were
quantified using qPCR to test a drug-gene combinatorial effect
(FIG. 58). Significant increase in the expression level of GM-CSF
was observed 3 days post transfection (.about.8,000 fold over the
control, ctrl) with PMAXGFP-GM-CSF (GMCSF). The addition of
DOPE/CHEMS (GMCSF+ DOPE/CHEMS) or SAHA (GMCSF+5 .mu.M SAHA)
enhanced GMCSF expression by .about.9000 and .about.14,000 fold,
respectively. Intriguingly, combination of DOPE/CHEMS and SAHA
elicited a far superior effect on GM-CSF overexpression
(.about.30,000 fold as compared to ctrl). These unanticipated
results are suggestive of a distinct function of SAHA beyond merely
stabilizing the tubulin network in enhancing GM-CSF expression. In
addition to the increment with GM-CSF expression with TrafEn.TM.,
SAHA induced up-regulation of MICA and NKG2D up to 48 hours of
treatment (FIG. 59). This observation is in line with the report on
the effect of SAHA on multiple cell lines. Increased expression of
MICA and NKG2D were responsible for the activation of NK cells.
Together, these data suggest the TrafEn.TM. gene-drug combination
as a promising strategy to generate cancer vaccine that activates
both APC (GM-CSF) and NK cells (MICA).
Example 17
Synergistic Effect of OSKM Overexpression and VPA/Tubastatin A to
Increase Reprogramming Efficiency of Human Fibroblast
[0157] Reprogramming describes the process of cellular state
conversions, including switch of differentiated cells into a less
differentiated state. Genetic tools have been established to
initiate reprogramming process in various cell types. For instance,
the Yamanaka et al. successfully identified four transfection
factors, Oct4, Sox2, Klf4, and cMyc, sufficiently generated iPSCs
from fibroblast. There are several concerns in the clinical use of
reprogrammed cell sources. The use of viral vectors may introduce
risk of tumourigenesis and immunogenesis. Another concern is the
risk of tumourigenesis induced by transgene integration. In
particular, overexpression of c-Myc, a well-known oncogene, and its
reactivation could cause tumour formation. Removal of c-Myc from
the reprogramming cocktail greatly reduces the reprogramming
efficiency. Interestingly, the functions of both cMyc and Klf4 can
be compensated by treating cells with HDACi such as Valproic acid
(VPA).
[0158] High N/P ratio of PEIMAX is required for transfection of
human adult fibroblast (HaF) (FIG. 60). Increased N/P (from 50-70)
did not result in further improvement of transfection suggest the
presence of intracellular transfection barrier. Addition of
TrafEn.TM. (PEIMAX-N/P=50, DOPE/CHEMS+1.0 .mu.M Tubastatin A)
resulted in drastic increment in the percentage of cells expressed
GFP, .about.70%. Conversely, transfection event was not detected
with Lipofectamine and Satisfection mediated transfection (FIG.
61A). Using the TrafEn.TM. formulation to deliver the polycistronic
expression vector (Addgene: 20328), expression levels of cMyc,
KLF4, SOX2, OCT4 were found to be .about.4000, .about.4500,
.about.4800 and .about.5400 fold higher as compared to the control
(PMAXGFP). In the absence of TrafEn.TM., OSKM levels were
.about.1000 fold as compared to the control (FIG. 61B). The high
level of expressions may contribute to the rapid formation of iPSC
like colonies. Nine days after transfection, colonies were observed
in cell culture transfected with TrafEn.TM. galenics (FIG. 61C).
Meanwhile, the effect of VPA on the induction of the expressions of
cMyc and KLF4 was explored (FIG. 62). Gradual increment of the
expressions of both cMyc and KLF4 were observed up to 72 h. Taken
together, these data suggest the TrafEn.TM. gene-drug combination
as a promising strategy to induce efficient reprogramming process
in human adult fibroblast using non-viral based method.
Discussion
[0159] To our knowledge, this is the first report that demonstrated
an unexpectedly high efficiency in transfecting differentiated
neuronal cell-lines and primary cortical neurons using a non-viral
carrier. The rational strategy involved mild centrifugation of
aggregated polyplex, facilitating endosomal escape and enhancing
microtubule trafficking. The choice of matched native and
differentiated neuronal cell models and development of reliable
quantitative tools greatly facilitated the identification and
mitigation of the hitherto unrecognized transfection barriers in
differentiated neurons using a non-viral carrier.
[0160] Differentiated neurons are sensitive to physical stress and
alterations in cellular environment, making transfection of these
cells a significant challenge. By limiting exposure of the cells to
polyplex and by mild centrifugation, transfection efficiency and
cell viability were significantly increased. Mild centrifugation is
thought to deposit aggregated polyplex, which is formed in the high
salt media (FIGS. 13 and 38-43). It is likely that polyplex is
inherently unstable in the high salt media. The deposited
polyplexes on the substrate was not released at significant levels
into the media (FIG. 43), an observation incongruent to previous
reports. Thus, deposited polyplex may be taken up directly from
cell surface or the cell culture substrate, consistent with recent
results showing the critical involvements of endocytic processes in
neuronal migration, motility and adhesion to substrates. The
critical role of deposited aggregated polyplex in mediating
efficient transfection was discussed under the supplementary
section (FIGS. 13 and 38-43).
[0161] Considerable efforts have been made to better understand the
mechanisms of uptake and intracellular trafficking of non-viral
carriers. Uptake of LPEI-polyplex has been shown to involve
multiple pathways including caveolae and clathrin-mediated
endocytosis. The precise size limits of clathrin- and
caveolin-mediated endocytosis are currently unclear (120-500 nm).
Recent evidences demonstrated the uptake of even larger
LPEI-polyplex by a rottlerin-sensitive and unselective
macropinocytotic pathway resulting in successful transfection of
mammalian cells. Unexpectedly, the uptake of DNA in the native but
not differentiated neuronal cells was sensitivity to PKC,
indicative of alterations in the uptake pathway/s and intracellular
trafficking of polyplex, which may have contributed to the
differential localization of polyplex into acidic compartment.
[0162] In contrast to previous reports, the quantitative uptake of
DNA is comparable between native and differentiated neuronal cells.
Despite similar levels of uptake, differentiated neuronal cells
were poorly transfected. This finding is consistent with previous
reports, where attempts to increase transfection in differentiated
neurons by enhancing uptake using polymers modified with RGD, HIV
TAT, Tet-1, HGP have met with only modest improvements.
Intriguingly, a differential localization of polyplex was observed
between native and differentiated neuronal cells. Consistent with
previous reports, polyplex was sequestered in acidic compartment in
differentiated neurons. LPEI is thought to exert "proton sponge
effect" but LPEI-polyplex was found to be entrapped within the
acidic compartment on neuronal differentiation and the addition of
DOPE/CHEMS released the labelled DNA from this compartment. The
dramatic enhancement of endosomal escape of labelled pDNA and
transfection efficiency by DOPE/CHEMS is unexpected and is
consistent with the idea that endosomal escape of polyplex is
critical. The pH-dependent destabilization of DOPE/CHEMS within the
acidic endosomal environment is thought to result in membrane phase
transition resulting in membrane fusion and eventually releasing
the contents into the cytosol. It is worthy to note that other
commercial endosome targeting reagents including chloroquine have
no effect on transfection efficiency.
[0163] It has been shown that polyplex, similar to some viruses,
utilizes the microtubule network to traffic through cytoplasm to
the nucleus. Thus, stabilization of microtubule results in greater
recruitment of dynein and kinesin motors, which enhances
transfection. The capability to facilitate efficient endosomal
escape using fusogenic lipids along with stabilization of the
microtubule network serves as an attractive synergistic strategy
for enhancing polymer-mediated transfection. Indeed, synergistic
effect was observed when microtubule stabilization was induced.
Evidently, with this rational approach, high transfection
efficiency in differentiated neuronal cells and primary neurons has
now been achieved. Intriguingly, the effect of fusogenic lipid and
Tubastatin A enhanced transfection is synergistic and temporally
controlled, suggesting that intracellular trafficking of polyplex
is tightly controlled in differentiated neurons. Polyplex may be
required to be released in the early stages of endocytosis and
intracellularly trafficked to prevent degradation of DNA or
transportation of polyplex to a non-productive pathway.
[0164] By rationally mitigating the contributions of the barriers
using the combinatorial reagents and strategy described herein, it
may not be surprising that other cationic polymers and derivatives
may now be successfully used for neuronal transfection in vitro and
in vivo, by exploiting colloidal stable polyplex that promote
self-assembled supramolecular ensembles on cell membrane and
directed targeting of selective intracellular trafficking pathways.
In line with this suggestion, significant transfection enhancements
have been observed with other cationic polymers (e.g., dendrimers
and BPEI) and with non-neuronal cell types that are well known to
be recalcitrant to cationic polymer based gene delivery (FIG. 30,
FIG. 44 and FIG. 45). It is also worthy to note that the use of
fusogenic reagent and HDAC inhibitor did not cause long-term effect
on global cellular processes.
[0165] The elucidation of limiting barriers to transfection led to
a rational approach of highly efficient gene delivery into
differentiated neuronal cells in vitro. By re-routing the
endosomal-released polyplex using pH sensitive lipids and enhancing
microtubule mediated trafficking, high levels of transfection were
achieved in differentiated neuronal cells and primary cortical
neurons. These hitherto unrecognized changes in the trafficking of
aggregated polyplex may provide a possible explanation for the
differences in the transfection efficiencies of native and
differentiated neurons. Thus, this study provides useful insights
for the rational design of synthetic non-viral carriers and
optimized galenics for gene transfection using cationic polymers
into differentiated neuronal cells in vitro.
[0166] Fusogenic agents, for example
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/cholesteryl
hemisuccinate (CHEMS), improved gene delivery. Fusogenic agents
facilitate endosomal escape through the pH-dependent
destabilization of DOPE/CHEMS within the acidic endosomal
environment, resulting in membrane phase transition and membrane
fusion. As shown in further examples, DOPE/CHEM specifically
re-directs carrier/DNA complexes away from the non-productive
acidic compartment, thus leading to enhanced transfection
efficiency. Fusogenic agents with properties similar to DOPE
include Dipalmitoylphosphatidylcholine (DPPC) and
1,2-Dioleoyl-sn-glycero-3-Phosphatidylcholine (DOPC). Some peptides
were found to have fusogenic property as well, such as
haemagglutinin (HA2-peptide), influenza-derived fusogenic peptide
diINF-7, T domain of Diphtheria toxin and polycationic peptides,
such as polylysine and polyarginine.
[0167] The use of microtubule-targeting agent and epigenetic
modifiers as first- and second line treatments in patients with
various diseases is known in the art. The disclosure further
encompasses the use of microtubule-targeting agents and epigenetic
modifiers. Here, a microtubule-targeting chemosensitizer (Mt-C) is
categorized as tubulin binding agents (TBA) and histone deacetylase
inhibitors (HDACi) (FIG. 3).
[0168] The two major classes of tubulin binding agents are known in
the art, taxanes and epothilones. TBA can suppress microtubule
dynamics, leading to mitotic block and apoptosis. Taxanes may
include paclitaxel (Taxol), docetaxel (Taxotere) or combinations
thereof. 15, Epothilones, like taxanes, are known in the art to
prevent cell division by inducing cell cycle arrest at the G2-M
transition phase, resulting in cytotoxicity and, in due course,
cell death. Epothilone analogues known in the art are patupilone,
ixabepilone, BMS 310705, sagopilone, KOS-862, and KOS-1584.
[0169] Table 1 below shows HDACi which have an inhibitory effect on
HDAC/Sirtuin. HDACi, as known in the art, have been shown to have
microtubule-targeting and/or epigenetic modification activities.
HDACi inhibit HDAC6 and Sirtuin2 to promote acetylation of
microtubules, resulting in the stabilization of the microtubule
network. Acetylation has been repeatedly alleged to be associated
with stability of microtubules, where the phrase `acetylated
microtubules` is often used synonymously with `stable
microtubules`. Similar to the effect of TBA, HDACi are known to
arrest microtubule dynamics and exerts anti-cancer effect. Some
HDACi, like vorinostat, inhibit multiple HDAC (HDAC1, 2, 3 and 6)
and exhibit various activities, such as immunomodulation and
apoptosis. HDACs are involved in the maintenance and function of
chromatin via regulation of acetylation state of histone.
Additionally, it is known that HDACi are known to have diverse
functions, as the HDAC family influences a broad repertoire of
physiological processes, including transcription of genes involved
in proliferation, differentiation, survival and DNA repair. More
recently, HDACi have been demonstrated to promote self-renewal of
hematopoietic stem cells, enhance differentiation of neural stem
cells and increase efficiency of reprogramming of somatic
cells.
TABLE-US-00001 TABLE 1 HDACi with inhibitory effect on
HDAC/Sirtuin. Developmental status of the inhibitors on various
diseases was presented. Targeted Targeted Developmental class of
HDAC/ IC50 HDAC inhibitor Class Type of disease status HDAC Sirtuin
(uM) Apicidin Peptide NA Tool compound Class I 1 0.02 2 0.02 3 0.02
Belinostat HA Cutaneous T-cell Phase II clinical Class I, IIb 1
0.25 (PXD101) lymphoma 2 0.25 Acute myeloid leukemia Phase I/II 3
0.2 6 0.41 BML-210 Benzamide NA tool compound Class I 1 14.99 2
10.91 3 2.62 CHR-2845 HA Hematological disease Phase I Class I NA
NA Lymphoid malignancies Phase I Bufexamac Non- NA Approved drug
Class I, IIb 3 341 steroidal (Paraderm .RTM., 6 10.7 Parfenac
.RTM.) 8 235 10 12.3 Dacinostat HA NA Phase I Class I, IIb 1 0.01
(NVP-LAQ824) 2 0.02 3 0.1 6 0.23 10 0.58 Entinostat Benzamide
Refractory Hodgkin's Phase II Class I 1 6.23 (MS-275; SNDX-
lymphoma 2 8.31 275) ER+ breast cancer Phase II 3 4.56
immunosuppressive - Phase I 8 201.36 infectious disease
JNJ-26481585 HA Acute myeloid leukemia Phase I Class I NA NA
Precursor cell Phase I lymphoblastic leukemia- lymphoma BCR-ABL
positive Phase I chronic myelogenous leukemia MC-1293 HA NA tool
compound Class I 3 288 Mocetinostat Benzamide Follicular lymphoma
Phase II Class I 1 15.49 (MGCD-0103) 2 17.59 3 6.72 Panobinostat HA
Classical Hodgkin's Phase III Class I, IIb 1 0.1 (LBH-589) lymphoma
Relapsed/Refractory Phase II 2 0.13 Classical Hodgkin's lymphoma
Multiple myeloma Phase I 3 0.27 Primary myelofibrosis Phase II 6
1.29 Post-polycythemia vera Phase II 10 0.54 myelofibrosis
Post-essential Phase II thrombocytopenia myelofibrosis PCI-24781 HA
Soft tissue sarcoma Phase I/II clinical Class I, IIb 1 0.02 2 0.03
3 0.05 6 0.05 10 0.03 PCI-34051 HA Anti-cancer Pre-clinical Class I
8 2.3 Romidepsin Peptide Cutaneous T-cell Phase II Class I 1 0.08
(FK228; FR901228) lymphoma Approved 2 0.01 drug(Istodax .RTM.) 3
0.02 Resminostat Hydroxamate Hepatocellular carcinoma Phase II
Class I, II NA NA Hodgkin's lymphoma Phase I SAHA NA cutaneous
T-cell Phase I Class I, IIb 1 0.29 (vorinostat) lymphoma Refractory
large B-cell Phase II lymphoma Stage IIIA non-small cell Phase I/II
lung cancer, Stage IIIB non-small cell Phase I/II lung cancer
Multiple myeloma Phase III 2 0.37 Acute/chronic myeloid Phase I
leukemia Acute promyelocytic Phase I leukemia Myelodysplastic Phase
I syndromes Relapsed/Refractory Phase I 3 0.39 multiple myeloma
Acute myeloid leukemia Phase II Brain and central nervous Phase I
system tumor Lymphoma Phase I Unspecified childhood Phase I 6 0.21
solid tumor Sarcoma Phase II Relapsed/Refractory Phase II multiple
myeloma Carcinoma, non-small cell Phase II lung cancer Scriptaid HA
NA tool compound Class I. II 1 1.37 2 1.51 3 4.24 6 0.25
Tacedinaline Benzamide Multiple Myeloma and Phase II Class I 1
13.08 Plasma Cell Neoplasm 2 12.84 3 5.39 Trichostatin A HA
Systemic lupus Phase I Class I, IIb 1 0.006 erythematous 2 0.007
Rheumatoid arthritis Phase I 3 0.02 6 0.06 Valproic Acid Fatty acid
Neuroectodermal tumor Phase I Class I, IIb 1 442.18 Brain
metastases Approved 2 485 Advanced cancer drug(Depakote .RTM., 3
4177 Depakene .RTM., 6 11784 Depacon .RTM., Stavzor .RTM.) B2 NA
Neurodegenerative disease Pre-clinical Class III SIRT2 35 Salermide
Amide Anti-cancer Pre-clinical Class III SIRT1 NA SIRT2 Sirtinol
Amide Anti-cancer Pre-clinical Class III SIRT2 40 Grey code
represents possible microtubule stabilization effect of inhibitor
and its application in TrafEn .TM.-Gene combination strategy. HA:
hydroxamic acid.
[0170] HDAC6 inhibition is known to affect microtubule dynamics in
non-neuronal cells and neurite outgrowth in neuronal cells. Thus,
the possibility of long-term effect of Tubastatin Aon cellular
processes of neuronal cells including microtubule dynamics,
neurogenesis and global cellular metabolism deserve careful
elucidation. Evidently, treatment with Tubastatin A results in
transient effect (<24 h) on tubulin acetylation (FIG. 35).
Moreover, the transfection paradigm described herein did not result
in sustained alteration on neurite outgrowth (FIG. 36) and global
metabolism (FIG. 37) of the neuronal cells.
[0171] MSC are a heterogeneous subset of adult stromal stem cells
that can be isolated and expanded ex vivo, and can appropriately
differentiate into cells of the residing tissues, repair the
damaged tissues and restore normal functions. Other than their
differentiation potential to osteogenic, chondrogenic, and
adipogenic cell types, studies have demonstrated the capability of
MSC to generate neurons, kidney and other cell types too. Viral
mediated delivery of BMP2 expression vector have been developed to
drive such stem cells into osteogenic cells by providing genetic
signals to improve the outcome of differentiation protocol. In
addition to the use of MSC as a cell source to generate
differentiating cells, it has been tested to act as gene/drug
delivery vehicles. MSCs were genetically modified with BMP2 to
facilitate tissue repair. On the other hand, an emerging approach
to enhance differentiation of MSC is the direct treatment with
HDACi. It has been suggested in numerous reports that HDACi such as
TSA, SAHA and valproic acid can promote osteogenic differentiation
of MSC. These HDACi induced epigenetic alterations resulting in the
up-regulations of osteogenic promoting factors such as BMP2, RUNX,
osterix, and osteopontin. To date, the synergistic effect of BMP2
transfection and TSA has yet to be tested.
Methods
[0172] Cell Culture
[0173] Neuro2A (ATCC: CCL-131TM) stably expressed GFR.alpha.2a
cells and rat primary cortical neurons' were cultured and
maintained as described previously (Yoong, L. F., G. Wan, and H. P.
Too. Mol Cell Neurosci, 2009. 41(4): p. 464-73; Zhou, L. and H. P.
Too. PLoS One, 2011. 6(6): p. e21680). Non-neuronal cells comprise
<0.5% of the cell population of neurons grow in Neurobasal-B27
(Brewer, G. J., et al. J Neurosci Res, 1993. 35(5): p. 567-76). On
DIV 3 (3 days in vitro), primary neurons were transfected. For
transfection of differentiated neuronal cells, Neuro2A and NG-108
cells were differentiated with 50 ng/ml glial cell-line derived
neurotrophic factor (GDNF; Biosource, Camarillo, Calif.), 10 .mu.M
all trans retinoic acid (RA) or 10 .mu.M Forskolin (FSK; Sigma, St.
Louis, Mo.) in Dulbecco's Modified Eagle Media (DMEM) supplemented
with 1% Fetal Bovine Serum (FBS) for 48 h prior transfection.
[0174] Generation of TrafEn.TM.
[0175] Firstly, the lipid components (e.g. DOPE and CHEMS) were
dissolved in chloroform. These components were mixed at a certain
ratio (DOPE:CHEMS), depending on the intended applications and type
of lipid used to form the fusogenic liposome. The solvent was then
evaporated, thus facilitating the formation of a lipid film
comprising DOPE/CHEMS at the bottom of glass tube. The lipid film
was reconstituted in 25 mM HEPES buffer by vigorous shaking, after
which the lipid solution was sonicated for 2 min in a bath-type
sonicator. This lipid solution construes the first agent. The
TrafEn.TM. composition is then prepared by combining the first
agent and a second agent, as defined in the disclosure. The
TrafEn.TM. composition referenced also envisions the usage of
fusogenic peptides instead of fusogenic liposomes.
[0176] Transfection Procedure
[0177] Plasmid DNA expressing EGFP was purified according to
manufacturer's instruction (Geneaid Biotech, Taiwan). LPEI (25-kDa;
Polyscience, USA) was added to pDNA in 25 mM HEPES buffer at
different N/P ratios and incubated at room temperature for 15 min.
LPEI/pDNA complex was then added to complete media (1:10) to
prepare the transfection mixture (pDNA at 2 .mu.g/ml). For bulk
transfection (with or without centrifugation), cells were seeded 24
h prior to transfection. The transfection mixture was added to
cells in culture and centrifuged at 280 g for 5 min. The
transfection mixture was then replaced with complete media and the
cells were further incubated. For transfection of differentiated
cells, transfection mixture was replaced with DMEM (1% FBS)
containing corresponding differentiation reagent. Transfection
efficiency (percentage of EGFP positive cells) was quantified,
either through manual counting or fluorescence-activated cell
sorting (FACS) analysis after incubation for indicated periods.
[0178] For studies using inhibitors, cells were incubated with
Dynasore (30 .mu.g/ml), Filipin III (5 .mu.g/ml) or Rottlerin (2.5
.mu.g/ml) in DMEM (0.5% FBS) for 45 min prior transfection. Cells
were further incubated in DMEM (0.5% FBS) containing corresponding
inhibitors post transfection. At the concentrations used, there was
no evidence of cell death.
[0179] To improve transfection in differentiated neurons,
DOPE/CHEMS and Tubastatin A (Bio Vision, San Francisco, USA) were
used. Lipid film comprising DOPE/CHEMS (9:2 molar ratio) (Polar
Avanti Lipid, Alabaster), was formed at the bottom of glass tube
after evaporation of the solvent, chloroform. The lipid film was
reconstituted in 25 mM HEPES buffer and sonicated for 2 min in a
bath-type sonicator (Brandson 2200). The lipid solution was added
to the cell culture at various indicated time post transfection.
One hour post transfection, Tubastatin A (5 or 16 .mu.M) was added
to the culture media. The inhibitor containing media were replaced
by fresh media 24 h later.
[0180] Flow Cytometry Analysis
[0181] After transfection, the cells were trypsinized, centrifuged
and re-suspended in PBS. Cell clumps were removed by filtering
through a 40 .mu.m mesh. The percentage of cells expressing EGFP
was quantified by FACS analysis (BD FACSCanto, BD Biosciences) and
the raw data analyzed using WinMDI (V2.9). At least 10,000 cells
were analysed per sample.
[0182] Real-Time qPCR for Quantification of DNA
[0183] To quantify DNA internalized by the cells, the supernatant
was removed and the cells were washed once with 1.times.PBS and
incubated in DMEM containing pAA/DNAse for 2 h at 4.degree. C.
Next, the cells were trypsinized and treated with pAA/urea lysis
buffer. Efficiency of DNA release by pAA/urea lysis buffer and
pAA/DNase was evaluated (Supplementary FIGS. 15 & 18).
Quantification of DNA after incubation of Trypsin with deposited
polyplex alone revealed insignificant amount of pDNA detected in
the trypsin fraction (data not shown). Primers specific for EGFP,
forward primer (5'-3', GACCACTACCAGCAGAACACC) and reverse primer
(5'-3', GACCATGTGATCGCGCTT) were used. PCR quantification of the
pDNA was performed as previously described (Yoong, L. F., G. Wan,
and H. P. Too. Mol Cell Neurosci, 2009. 41(4): p. 464-73). The
absolute amount of pDNA was determined using plasmid standards by
interpolation.
[0184] Imaging Studies
[0185] FITC- and Rhodamine-pDNA were prepared according to
manufacturer recommendation (Minis Bio, USA). Expression of EGFP
was observed in cells transfected with fluorescently labeled pDNA
(data not shown). FITC- or Rhodamine-pDNA was used for
visualization of internalized polyplex and extracellular
fluorescence of the labeled pDNA was quenched with EtBr (20
.mu.g/ml) or 0.4% trypan blue respectively 4 h post transfection.
Cell images were taken before and after quenching with an inverted
Zeiss microscope equipped with fluorescence detection (Zeiss cell
observer Z1) and processed (Axio Vision Rel. 4.7). Quenching
efficiency of trypan blue was examined (Supplementary FIG. 7). To
study co-localization of Rhodamine-pDNA and the acidic compartment,
cells were incubated with lysotracker green DND-26 (50 nM) for 5
min Image was captured with a Zeiss confocal microscopy (LSM710,
Oberkochen, Germany). Co-localized pixel was analysed with Zeiss
ZEN software (v2010).
[0186] Cell Viability Assay
[0187] To access the influence of LPEI mediated transfection on
cellular viability of native and differentiated neuronal cells,
Neuro2A and NG-108 cells were differentiated with GDNF (50 ng/ml)
or RA (10 .mu.M) and Fsk (10 .mu.M) in the 96-well plates 48 h
prior transfection. Cells were then exposed to LPEI/pDNA (at
various N/P ratios) for 15 min or 4 h. With or without
centrifugation at 280 g for 5 min, transfection mixtures were
replaced with complete medium and incubated for 48 h. Culture
medium was replaced with 100 .mu.l DMEM and 20 .mu.l of CellTiter
96 Aqueous One Solution Cell Proliferation Assay (Promega,
Singapore). Following 1 h incubation at 37.degree., the absorbance
at 490 nm was recorded using a 96-well Microplate reader (Model
680, Biorad).
[0188] DNA Release Assay
[0189] The DNA was collected through the following procedures: (1)
to quantify DNA in supernatant, the supernatant was treated with
pAA/urea lysis buffer to release DNA; (2) to quantify DNA
associated with cells, the supernatant was removed and the cells
were washed once with 1.times.PBS before being harvested by
trypsinization and treated with pAA/urea lysis buffer; (3) to
quantify DNA internalized by the cells, the supernatant was removed
and the cells were washed once with 1.times.PBS and incubated in
DMEM containing pAA/DNAse for 2 h at 4.degree. C. Next, the cells
were trypsinized and treated at 95.degree. C. for 30 min with
pAA/urea lysis buffer-polyacrylic acid (pAA, Sigma, Mw: 8000; 10 ng
of pAA/ng of pDNA; 32 carboxyl groups in pAA/1 phosphate group in
pDNA), 0.5 M sodium chloride, 10 mM sodium phosphate and 4 M urea.
pAA was used as a competitive reagent for displacement of pDNA from
LPEI. The efficiency of pDNA dissociation was visualized/quantified
using two different approaches--DNA retardation assay and qPCR. DNA
complexes were electrophoresed (100 V, 20 min) in 0.8% agarose gel,
stained with ethidium bromide and visualized on a UV
transilluminator.
[0190] Removal of Surface Bound pDNA
[0191] After transfection, cells (in 6-well plate) were incubated
with 400 .mu.l serum free DMEM containing pAA (10 ng pAA/ng pDNA),
10 mM CaCl.sub.2, 6 mM MgCl.sub.2 and 4 unit/ml deoxyribonuclease I
(DNAse) for 2 h at 4.degree. C. This formulation was optimized for
complete release and degradation of surface bound pDNA. Plasmid DNA
complexed with LPEI was displaced by pAA and degraded by DNAse I in
the presence of CaCl.sub.2 and MgCl.sub.2. Incubation at 4.degree.
C. prevented internalization of DNAse by the cells. RT-qPCR and
imaging studies confirmed the efficient removal of surface bound
pDNA using this approach (Supplementary FIG. 18).
[0192] Dynamic Light Scattering
[0193] LPEI/pDNA (N/P=20) were prepared in 25 mM HEPES or DMEM.
Particle size was measured by dynamic light scattering using
Zetasizer Nano (Malvern, Worcestershire, United States).
[0194] Deposit Mediated Transfection
[0195] For deposit-mediated transfection, the transfection mixture
was transferred to the culture plate (pre-coated with complete
media for 24 h) and incubated for the indicated periods (with or
without centrifugation). The transfection mixture was removed and
cells were seeded. Transfection efficiency (percentage of EGFP
positive cells) was quantified, either through manual counting or
through FACS analysis 48 h post transfection.
[0196] Immunocytochemistry
[0197] Control and 10 .mu.M differentiated Neuro2a Cells were fixed
with 4% formaldehyde in 1.times.PBS for 20 min at room temperature
and subsequently permeabilized in 0.5% Triton-X100 in 1.times.PBS.
Then, the samples fixed cells were blocked with normal goat serum
(1:10; Dako, Glostrup, Denmark) in 0.1% Triton X-100/1.times.PBS
for 30 min at 37.degree. C. The cells were then incubated with
primary antibodies against acetylated .alpha.-Tubulin (Sigma
Aldrich T7451, 1:200 dilution) or TuJ (R&D Systems MAB1195,
1:50 dilution) in 0.1% TritonX-100/1% BSA/1.times.PBS at 37.degree.
C. for 2 h and washed three times in 1.times.PBS. Subsequently, the
cells were incubated with goat anti-mouse fluorescent secondary
antibody (AlexaFluor 488/596; Invitrogen, CA) diluted 1:200 in 0.1%
Triton X-100/1% BSA/1.times.PBS for 2 h at 37.degree. C. The cells
were washed three times in 1.times.PBS and mounted. Image
acquisition was performed using the Zeiss LSM710 with Axio
Observer.Z1 confocal microscope system (Oberkochen, Germany). All
images were taken with identical laser and optical settings.
[0198] Metabolites Assay by LC-MS
[0199] Cells (cultured in 6-well plate, .about.1 million cells)
were rapidly rinsed by 2 mL of ice cold HPLC water. Then, the
metabolic states of the cells were quenched by subjecting the
culture plate to liquid nitrogen after aspiration of the HPLC
water. After 1 min incubation, 250 .mu.L of ice cold
methanol:chloroform (9:1 ratio) was added to each well for
extraction purpose and cells were scraped with a cell scraper
(Greenpia Tech.) (Lorenz, M. A., C. F. Burant, and R. T. Kennedy.
Anal Chem, 2011. 83(9): p. 3406-14). Extracts were transferred to
1.5 mL micro-centrifuge tubes containing 0.1 mm glass beads
(Biospec Product). To release the metabolites efficiently, the
samples were subjected to Mini-Beadbeater (Bio Spec Products Inc.)
for 2 min. After which, the extracts were pelleted by
centrifugation for 3 min at 16 000 g. The supernatants were
transferred to autosampler vials (Agilent tech.) and assayed.
[0200] The UPLC (Waters ACQUITY UPLC)--(TOF) MS (Bruker micrOTOF
II) platform was used to analyse the metabolites. To scan 50-800
m/z in negative mode with -500 V end plate voltage and 4500 V
capillary voltage, electrospray ionization was used and (TOF) mass
spectrometry was operated. One bar of nebulizer gas was provided.
The drying gas rate and dry gas temperature was adjusted at 9
mL/min and 200.degree. C. respectively. From the acquired data
(50-800 m/z), a range of m/z (0.06 m/z width, the average m/z
distribution width with the MS instrument in use) was extracted).
Subsequently, the retention time was determined for each
intermediate and the peak area was integrated for each metabolite
using manufacturer's software.
[0201] Relative amount of each metabolite to ATP was calculated.
ATP was used as normalizer as it was the most abundant metabolite
and stable across samples at each time point with coefficient
variation less than 5%. After normalization, relative fold change
of metabolite to the negative control at each time point was
calculated.
Sequence CWU 1
1
2121DNAArtificial SequenceA synthetic oligonucleotide 1gaccactacc
agcagaacac c 21218DNAArtificial SequenceA synthetic oligonucleotide
2gaccatgtga tcgcgctt 18
* * * * *